Binding polypeptides and methods based thereon

ABSTRACT

Binding polypeptides that specifically bind BLyS protein or BLyS-like polypeptides can be used in methods of the invention for detecting, diagnosing, or prognosing a disease or disorder associated with aberrant BLyS or BLyS receptor expression or inappropriate function of BLyS or BLyS receptor, comprising BLyS binding polypeptides or fragments or variants thereof, that specifically bind to BLyS. The present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS function or BLyS receptor function, comprising administering to an animal, preferably a human, an effective amount of one or more BLyS binding polypeptides or fragments or variants thereof, that specifically bind to BLyS.

FIELD OF THE INVENTION

The present invention relates to therapeutic and diagnostic uses formolecules that bind to B lymphocyte stimulator protein (BLyS). Inparticular, the present invention also relates to methods andcompositions for detecting, diagnosing, or prognosing a disease ordisorder associated with aberrant BLyS or BLyS receptor expression orinappropriate function of BLyS or BLyS receptor, comprising BLyS bindingpolypeptides or fragments or variants thereof, that specifically bind toBLyS. The present invention further relates to methods and compositionsfor preventing, treating or ameliorating a disease or disorderassociated with aberrant BLyS or BLyS receptor expression orinappropriate BLyS function or BLyS receptor function, comprisingadministering to an animal, preferably a human, an effective amount ofone or more BLyS binding polypeptides or fragments or variants thereof,that specifically bind to BLyS.

BACKGROUND OF THE INVENTION

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor(“TNF”) superfamily that induces both in vivo and in vitro B cellproliferation and differentiation (Moore et al., Science, 285: 260-263(1999)). BLyS is distinguishable from other B cell growth anddifferentiation factors such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13,IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and proteinexpression pattern and its specific receptor distribution and biologicalactivity on B lymphocytes. BLyS expression is not detected on naturalkiller (“NK”) cells, T cells or B cells, but is restricted to cells ofmyeloid origin. BLyS expression on resting monocytes is upregulated byinterferon-gamma (IFN-gamma). The gene encoding BLyS has been mapped tochromosome 13q34.

BLyS is expressed as a 285 amino acid type II membrane-bound polypeptideand a soluble 152 amino acid polypeptide (Moore et al., 1999, supra).The membrane-bound form of BLyS has a predicted transmembrane spanningdomain between amino acid residues 47 and 73. The NH₂-terminus of thesoluble form of BLyS begins at Ala¹³⁴ of the membrane-bound form ofBLyS. Both the soluble and membrane-bound forms of the protein formhomotrimers. Soluble recombinant BLyS has been shown to induce in vitroproliferation of murine splenic B cells and to bind to a cell-surfacereceptor on these cells (Moore et al., 1999, supra). Soluble BLySadministration to mice has been shown to result in an increase in theproportion of CD45R^(dull), Ly6 D^(bright) (also known as ThB) B cellsand an increase in serum IgM and IgA levels (Moore et al., 1999, supra).Thus, BLyS displays a B cell tropism in both its receptor distributionand biological activity.

Based on its expression pattern and biological activity, BLyS has beensuggested to be involved in the exchange of signals between B cells andmonocytes or their differentiated progeny. The restricted expressionpatterns of BLyS receptor and ligand suggest that BLyS may function as aregulator of T cell-independent responses in a manner analogous to thatof CD40 and CD40L in T cell-dependent antigen activation.

Accordingly, molecules that specifically bind BLyS would find a varietyof uses in the study of the BLyS cytokine, in the manufacture andpurification of BLyS in commercial and medically pure quantities, and inthe development new therapeutic or diagnostic reagents. BLyS bindingpolypeptides may also find medical utility in, for example, thetreatment of B cell and/or monocyte disorders associated withautoimmunity, neoplasia, or immunodeficiency syndromes.

SUMMARY OF THE INVENTION

New polypeptides that specifically bind to B lymphocyte stimulatorprotein (BLyS) and/or BLyS-like polypeptides have been discovered, andthe therapeutic and diagnostic applications for such polypeptides aredisclosed herein. Particular polypeptides useful in the methods of thisinvention specifically bind to a polypeptide or polypeptide fragment ofhuman BLyS (SEQ ID NOs:173 and/or 174) or BLyS expressed on humanmonocytes; murine BLyS (SEQ ID NOs:175 and/or 176) or BLyS expressed onmurine monocytes; rat BLyS (either the soluble forms as given in SEQ IDNOs:177, 178, 179 and/or 180 or in a membrane associated form, e.g., onthe surface of rat monocytes); or monkey BLyS (e.g., the monkey BLySpolypeptides of SEQ ID NOS:181 and/or 182, the soluble form of monkeyBLyS, or BLyS expressed on monkey monocytes), preferably human BLyS.

In preferred methods of the invention, BLyS binding polypeptidescomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-12, 20-172, and186-444, preferably SEQ ID NOs:163-172 and 436-444 as referred to hereinand in Tables 1-8, 13 and 14, and fragments and variants thereof, willbe used.

In specific preferred embodiments, the BLyS binding polypeptides bindBLyS and/or BLyS-like polypeptides with high affinity. In otherembodiments, the BLyS binding polypeptides reversibly bind BLyS and/orBLyS-like polypeptides. In still other embodiments, the BLyS bindingpolypeptides irreversibly bind BLyS and/or BLyS-like polypeptides.

The cysteine residues in certain polypeptides useful in the methods ofthe invention are believed to form a disulfide bond, which would causethe polypeptide containing these cysteine residues to form a stable loopstructure under non-reducing conditions. Especially preferred BLySbinding polypeptides useful in the methods of the invention arepolypeptide molecules that comprise amino acid sequences that formstable loop structures or other stable structures that bind BLyS orBLyS-like polypeptides.

Analysis of the sequences of the BLyS binding polypeptides describedherein shows a strong selection for polypeptides containing thetetrapeptide Asp-Xaa-Leu-Thr (SEQ ID NO:446), and therefore in itsbroadest aspects, the present invention relates to methods for usingpolypeptides capable of binding to BLyS comprising the polypeptideAsp-Xaa-Leu-Thr (SEQ ID NO:446), where Xaa is Pro, Ser, Thr, Phe, Leu,Tyr, Cys, or Ala (preferably Pro or Ser).

In addition, seven consensus sequences (SEQ ID NOs:1-7) are disclosedfor peptides useful in the methods of the invention, based on thespecific BLyS binding polypeptides shown in Tables 1-8. In preferredmethods according to the invention, BLyS binding polypeptides comprisingone or more of these sequences are used. Such preferred methods utilizeBLyS binding polypeptides including polypeptides with the potential toform a cyclic or loop structure between invariant Cys residuescomprising, or alternatively consisting of, an amino acid sequenceselected from A-E (SEQ ID NOs:1-5): (A)X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃, (SEQ ID NO:1)whereinX₁ is Ala, Asn, Lys, or Ser;X₂ is Ala, Glu, Met, Ser, or Val;X₃ is Ala, Asn, Lys, or Pro (preferably Lys);X₅ is Phe, Trp, or Tyr (preferably Tyr);X₇ is Pro or Tyr (preferably Pro);X₁₁ is Ala, Gln, His, Phe, or Val;X₁₂ is Asn, Gln, Gly, His, Ser, or Val; andX₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser,

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄, (SEQ ID NO:2)whereinX₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, Val, or is absent;X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val;X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Trp, Tyr, or Val (preferably Asp);X₅ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val(preferably Glu or Leu);X₇ is His, Leu, Lys, or Phe (preferably His or Leu);X₈ is Leu, Pro, or Thr (preferably Thr or Pro);X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys);X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, orVal;X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val;X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val; andX₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro,Trp, Tyr, Val, or is absent,

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅, (SEQ ID NO:3)whereinX₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr;X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp;X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr (preferably Ala);X₅ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);X₇ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser);X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val (preferably Val);X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; andX₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr (preferably Glu orPro),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆, (SEQ IDNO:4)whereinX₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent (preferablySer);X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably Arg);X₃ is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp);X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;X₇ is Asp, His, Leu, or Ser (preferably Asp);X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);X₉ is Ala, Arg, Asn, or Leu (preferably Leu);X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val;X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu);X₁₅ is Asn, His, Leu, Pro, or Tyr (preferably His, Leu or Pro); andX₁₆ is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp or Ser),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈,(SEQ ID NO:5)whereinX₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent(preferably Arg);X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent (preferably Asn,Asp, Gly, or Pro);X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val (preferably Glyor Met);X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val(preferably Trp, Tyr, or Val);X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferablyAsp);X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);X₉ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr(preferably Lys or Thr);X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferablyArg or Leu);X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr(preferably Thr or Trp);X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr(preferably Met or Phe);X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, orVal (preferably Val);X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp (preferablyMet);X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr, (preferablyArg, His, or Tyr); andX₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp (preferably His orAsn),wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

Additional preferred embodiments include methods utilizing linear BLySbinding polypeptides comprising, or alternatively consisting of, anamino acid sequence selected from F and G (SEQ ID NOs:6 and 7): (F)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂, (SEQ ID NO:6)whereinX₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val(preferably Gly, Tyr, or Val);X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr (preferablyHis or Tyr);X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably Asp or Tyr);X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val (preferablyAsp or Gln);X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably Leu or Ser);X₆ is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu or Thr);X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably Lys or Thr);X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr (preferably Leu or Lys);X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val(preferably Met or Ser);X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably Thr orLeu);X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, orVal (preferably Pro or Thr); andX₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val(preferably Arg or Pro),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃, (SEQ ID NO:7)whereinX₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp (preferably Glu, Lys);X₂ is Arg, Gln, His, Ile, Leu, or Pro (preferably His or Pro);X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably Tyr);X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr (preferably Asp or Gln);X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val (preferably Asn orThr);X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val (preferably Asp or Pro);X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr (preferably Ile orPro);X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or Thr);X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr (preferably Lys);X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably Gly or Met);X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val(preferably Ala or Thr);X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg orHis); andX₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val (preferably His),wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

Additional polypeptides useful in the methods of the invention includepolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from H-L (SEQ ID NOs:8-12): (H)Cys-X₂-Phe-X₄-Trp-Glu-Cys, (SEQ ID NO:8)whereinX₂ is Phe, Trp, or Tyr (preferably Tyr); and

X₄ is Pro or Tyr (preferably Pro); or (I) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys,(SEQ ID NO:9)whereinX₂ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);X₃ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val(preferably Glu or Leu);X₄ is His, Leu, Lys, or Phe (preferably His or Leu);X₅ is Leu, Pro, or Thr (preferably Thr or Pro);X₆ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys); and

X₇ is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr,or Val; or (J) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys, (SEQ ID NO:10)whereinX₂ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);X₃ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);X₄ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);X₅ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);X₆ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); or (K)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys, (SEQ ID NO:11)whereinX₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;X₃ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;X₄ is Asp, His, Leu, or Ser (preferably Asp);X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);X₆ is Ala, Arg, Asn, or Leu (preferably Leu);X₇ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);X₈ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

X₉ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; or (L)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys, (SEQ ID NO:12)whereinX₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val(preferably Trp, Tyr, or Val);X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferablyAsp);X₄ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);X₆ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr(preferably Lys or Thr);X₈ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferablyArg or Leu);X₉ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr(preferably Thr or Trp);X₁₀ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr(preferably Met or Phe);X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, orVal (preferably Val);wherein said polypeptides bind BLyS and/or BLyS-like polypeptides.

In preferred embodiments of the present invention, BLyS bindingpolypeptides are used which comprise the following amino acid sequence M(SEQ ID NO:447): (M) Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄,(SEQ ID NO:447)whereinX₂ is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu, Met, Thr,Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid except Arg;preferably Asn);X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);X₄ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);X₉ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp);X₁₂ is Leu, Phe, Val, Ile, or His (preferably Leu);X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe,or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or Trp; preferably Pro); andX₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys,Ile, Thr or Leu (i.e., not Phe, Met, Pro, or Trp; preferably Asp, Val orGlu).

Preferred methods will utilize polypeptides comprising a core sequenceof the formula N: (N) X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀, (SEQ IDNO:448)whereinX₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);X₂ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);X₇ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp); andX₁₀ is Leu, Phe, Val, Ile, or His (preferably Leu).

Especially preferred methods according to the invention will utilizeBLyS binding polypeptides which comprise the core peptideTrp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO:436).

BLyS binding polypeptides used in the methods of the present inventionmay also have an amino terminal (N-terminal) capping or functionalgroup, such as an acetyl group, which, for example, blocks the aminoterminal amino group from undesirable reactions or is useful in linkingthe BLyS binding polypeptide to another molecule, matrix, resin, orsolid support. BLyS binding polypeptides may also have a carboxyterminal (C-terminal) capping or functional group, such as an amidegroup, which, for example, blocks the C-terminal carboxyl group fromundesirable reactions or provides a functional group useful inconjugating the binding polypeptide to other molecules, matrices,resins, or solid supports. Preferably, the N- and/or C-terminal cappinggroups are polypeptide linker molecules. An especially preferredC-terminal linker molecule that is useful for immobilizing a BLySbinding polypeptide to a solid support or chromatographic matrixmaterial comprises the amino acid sequencePro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:13). Another usefulC-terminal linker, e.g., for fluoresceinating peptides, is Gly-Gly-Lys(see Table 14).

In the methods of the present invention, it may be advantageous to useBLyS binding polypeptides that have been modified, for example, toincrease or decrease the stability of the molecule, while retaining theability to bind BLyS and/or BLyS-like polypeptides. An example of amodified BLyS binding polypeptide is a polypeptide in which one of twocysteine residues is substituted with a non-naturally occurring aminoacid that is capable of condensing with the remaining cysteine sidechain to form a stable thioether bridge, thereby generating a cyclicBLyS binding polypeptide. Such cyclic thioether molecules of syntheticpeptides may be routinely generated using techniques known in the art,e.g., as described in PCT publication WO 97/46251, incorporated hereinby reference.

Some of the methods provided herein utilize BLyS binding polypeptidesthat have been attached, coupled, linked or adhered to a matrix or resinor solid support. Techniques for attaching, linking or adheringpolypeptides to matrices, resins and solid supports are well known inthe art. Suitable matrices, resins or solid supports for these materialsmay be any composition known in the art to which a BLyS bindingpolypeptide could be attached, coupled, linked, or adhered, includingbut not limited to, a chromatographic resin or matrix, such asSEPHAROSE-4 FF agarose beads, the wall or floor of a well in a plasticmicrotiter dish, such as used in an enzyme-liked immunosorbent assay(ELISA), or a silica based biochip. Materials useful as solid supportson which to immobilize binding polypeptides for use in the methodsinclude, but are not limited to, polyacrylamide, agarose, silica,nitrocellulose, paper, plastic, nylon, metal, and combinations thereof.A BLyS binding polypeptide may be immobilized on a matrix, resin orsolid support material by a non-covalent association or by covalentbonding, using techniques known in the art.

In certain embodiments of the present invention, it is preferred toutilize BLyS binding polypeptides or phage displaying such bindingpolypeptides that irreversibly bind the BLyS protein in its native,soluble trimeric form.

In certain embodiments of the present, it is preferred to utilize BLySbinding polypeptides of the present invention or phage displaying suchbinding polypeptides that reversibly bind the BLyS protein in itsnative, soluble trimeric form.

In further embodiments of the present invention, a method may call forthe use of a composition of matter comprising isolated nucleic acids,preferably DNA, encoding a BLyS binding polypeptide. In specificembodiments, nucleic acid molecules encode a BLyS binding polypeptidecomprising the amino acid sequence of SEQ ID NOs:1-12, 20-172, or186-444. In additional embodiments, the nucleic acid molecules encode apolypeptide variant or fragment of a polypeptide comprising an aminoacid sequence of SEQ ID NOs:1-12, 20-172, or 186-444. In a furtheradditional embodiment, such nucleic acid molecules encode a BLyS bindingpolypeptide, the complementary strand of which nucleic acid hybridizesto a polynucleotide sequence encoding a polypeptide described in Tables1-8 and 13 and in Examples 2, 5 and 6 (SEQ ID NOs:1-12, 20-172 and186-444), under stringent conditions, e.g., hybridization tofilter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45°C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65°C., under highly stringent conditions, e.g., hybridization tofilter-bound nucleic acid in 6×SSC at about 45° C. followed by one ormore washes in 0.1×SSC/0.2% SDS at about 68° C., or under otherstringent hybridization conditions which are known to those of skill inthe art (see, for example, Ausubel, F. M. et al., eds., 1989, CurrentProtocols in Molecular Biology, Vol. 1, Green Publishing Associates,Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and2.10.3).

In further embodiments of the invention, recombinant bacteriophage areutilized which display BLyS binding polypeptides on their surfaces. Suchphage may be routinely generated using techniques known in the art andare useful, for example, as screening reagents and reagents fordetecting BLyS.

In other methods according to the invention, a BLyS binding polypeptideis used to detect or isolate BLyS or BLyS-like polypeptides in asolution. Such solutions include, but are not limited to, BLyS orBLyS-like polypeptides suspended or dissolved in water or a buffersolution as well as any fluid and/or cell obtained from an individual,biological fluid, body tissue, body cell, cell line, tissue culture, orother source which may contain BLyS or BLyS-like polypeptides, such as,cell culture medium, cell extracts, and tissue homogenates. Biologicalfluids include, but are not limited to, sera, plasma, lymph, blood,blood fractions, urine, synovial fluid, spinal fluid, saliva, andmucous.

Methods according to the present invention may advantageously utilizepanels of BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants) wherein the panel members correspond to one, two, three, four,five, ten, fifteen, twenty, or more different BLyS binding polypeptides.Methods according to the present invention may alternatively usemixtures of BLyS binding polypeptides, wherein the mixture correspondsto one, two, three, four, five, ten, fifteen, twenty, or more differentBLyS binding polypeptides. The present invention also provides methodsof using compositions comprising, or alternatively consisting of, one,two, three, four, five, ten, fifteen, twenty, or more BLyS bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, BLyS binding polypeptide fragments or variants thereof).Alternatively, a method according to the invention may utilize acomposition comprising, or alternatively consisting of, nucleic acidmolecules encoding one or more BLyS binding polypeptides.

The methods of the present invention also provides for the use of fusionproteins comprising a BLyS binding polypeptide (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof), and a heterologous polypeptide. Acomposition useful in methods of the present invention may comprise, oralternatively consist of, one, two, three, four, five, ten, fifteen,twenty or more fusion proteins capable of binding to BLyS.Alternatively, a composition useful in methods of the invention maycomprise, or alternatively consist of, nucleic acid molecules encodingone, two, three, four, five, ten, fifteen, twenty or more such fusionproteins.

The present invention encompasses methods and compositions fordetecting, diagnosing, prognosing, and/or monitoring diseases ordisorders associated with aberrant BLyS or BLyS receptor expression orinappropriate BLyS or BLyS receptor function in an animal, preferably amammal, and most preferably a human, comprising, or alternativelyconsisting of, use of BLyS binding polypeptides (including moleculeswhich comprise, or alternatively consist of, BLyS binding polypeptidefragments or variants thereof) that specifically bind to BLyS. Diseasesand disorders which can be detected, diagnosed, prognosed and/ormonitored with the BLyS binding polypeptides include, but are notlimited to, immune system diseases or disorders (e.g., autoimmunediseases or disorders, immunodeficiencies, lupus, glomerular nephritis,rheumatoid arthritis, multiple sclerosis, graft vs. host disease,myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome),proliferative diseases or disorders (e.g., cancer, premalignantconditions, benign tumors, hyperproliferative disorders, benignproliferative disorders, leukemia, lymphoma, chronic lymphocyticleukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, Tcell proliferative diseases and disorders, B cell proliferative diseasesand disorders, monocytic proliferative diseases or disorders, acutemyelogenous leukemia, macrophage proliferative diseases and disorders,and carcinoma), infectious diseases (e.g., AIDS), and inflammatorydisorders (e.g., asthma, allergic disorders, and rheumatoid arthritis).

In specific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing and/or monitoringdiseases or disorders associated with hypergammaglobulinemia (e.g.,AIDS, autoimmune diseases, and some immunodeficiencies). In otherspecific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing and/or monitoringdiseases or disorders associated with hypogammaglobulinemia (e.g., animmunodeficiency).

The present invention further encompasses methods and compositions forpreventing, treating and/or ameliorating diseases or disordersassociated with aberrant BLyS or BLyS receptor expression orinappropriate BLyS or BLyS receptor function in an animal, preferably amammal, and most preferably a human, comprising, or alternativelyconsisting of, administering to an animal in which such treatment,prevention or amelioration is desired one or more BLyS bindingpolypeptides (including molecules which comprise, or alternativelyconsist of, BLyS binding polypeptide fragments or variants thereof) inan amount effective to treat, prevent or ameliorate the disease ordisorder. Diseases and disorders which can be prevented, treated, and/orameliorated with the BLyS binding polypeptides include, but are notlimited to, immune system diseases or disorders (e.g., autoimmunediseases or disorders, immunodeficiencies, lupus, glomerular nephritis,rheumatoid arthritis, multiple sclerosis, graft vs. host disease,myasthenia gravis, Hashimoto's disease, immunodeficiency syndrome,hypogammaglobulinemia, and hypergammaglobulinemia), proliferativediseases or disorders (e.g., cancer, premalignant conditions, benigntumors, hyperproliferative disorders, benign proliferative disorders,leukemia, lymphoma, chronic lymphocytic leukemia, multiple myeloma,Hodgkin's lymphoma, Hodgkin's disease, T cell proliferative diseases anddisorders, B cell proliferative diseases and disorders, monocyticproliferative diseases or disorders, acute myelogenous leukemia,macrophage proliferative diseases and disorders, and carcinoma),infectious diseases (e.g., AIDS), and inflammatory disorders (e.g.,asthma, allergic disorders, and rheumatoid arthritis).

In specific embodiments, the present invention encompasses methods andcompositions (e.g., BLyS binding polypeptides that antagonize BLySactivity) for preventing, treating and/or ameliorating diseases ordisorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmunediseases, and some immunodeficiency syndromes). In other specificembodiments, the present invention encompasses methods and compositions(e.g., BLyS binding polypeptides that enhance BLyS activity) forpreventing, treating or ameliorating diseases or disorders associatedwith hypogammaglobulinemia (e.g., an immunodeficiency syndrome).

In specific embodiments, the present invention encompasses methods andcompositions (e.g., BLyS binding polypeptides that antagonize BLySactivity) for preventing, treating and/or ameliorating immune systemdiseases or disorders, comprising, or alternatively consisting of,administering to an animal in which such treatment, prevention, and/oramelioration is desired, a BLyS binding polypeptide in an amounteffective to treat, prevent and/or ameliorate the disease or disorder.

In specific embodiments, the present invention encompasses methods andcompositions (e.g., BLyS binding polypeptides that antagonize BLySactivity) for preventing, treating and/or ameliorating diseases ordisorders of cells of hematopoietic origin, comprising, or alternativelyconsisting of, administering to an animal in which such treatment,prevention, and/or amelioration is desired, a BLyS binding polypeptidein an amount effective to treat, prevent and/or ameliorate the diseaseor disorder.

Autoimmune disorders, diseases, or conditions that may be detected,diagnosed, prognosed, monitored, treated, prevented, and/or amelioratedusing the BLyS binding polypeptides include, but are not limited to,autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmune neutropenia,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis,Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis,dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome,insulin dependent diabetes mellitis, and autoimmune inflammatory eye,autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis),systemic lupus erythematosus, discoid lupus, Goodpasture's syndrome,Pemphigus, Receptor autoimmunities such as, for example, (a) Graves'Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoidarthritis, schleroderma with anti-collagen antibodies, mixed connectivetissue disease, polymyositis/dermatomyositis, pernicious anemia,idiopathic Addison's disease, infertility, glomerulonephritis such asprimary glomerulonephritis and IgA nephropathy, bullous pemphigoid,Sjogren's syndrome, diabetes mellitus, and adrenergic drug resistance(including adrenergic drug resistance with asthma or cystic fibrosis),chronic active hepatitis, primary biliary cirrhosis, other endocrinegland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome,urticaria, atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulomatous, degenerative, and atrophic disorders).

Immunodeficiencies that may be detected, diagnosed, prognosed,monitored, treated, prevented, and/or ameliorated using the BLyS bindingpolypeptides include, but are not limited to, severe combinedimmunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminasedeficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton'sdisease, congenital agammaglobulinemia, X-linked infantileagammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

The present invention further encompasses methods and compositions forinhibiting or reducing immunoglobulin production, comprising, oralternatively consisting of, contacting an effective amount of BLySbinding polypeptide with BLyS, wherein the effective amount of BLySbinding polypeptide inhibits or reduces BLyS mediated immunoglobulinproduction.

The present invention further encompasses methods and compositions forinhibiting or reducing immunoglobulin production, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a BLyS binding polypeptide in anamount effective to inhibit or reduce immunoglobulin production.

The present invention further encompasses methods and compositions forinhibiting or reducing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of BLySbinding polypeptide with BLyS, wherein the effective amount of BLySbinding polypeptide inhibits or reduces BLyS mediated B cellproliferation.

The present invention further encompasses methods and compositions forinhibiting or reducing B cell proliferation comprising, or alternativelyconsisting of, administering to an animal in which such inhibition orreduction is desired, a BLyS binding polypeptide in an amount effectiveto inhibit or reduce B cell proliferation.

The present invention further encompasses methods and compositions forinhibiting or reducing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of BLySbinding polypeptide with BLyS, wherein the effective amount of BLySbinding polypeptide inhibits or reduces BLyS mediated B cell activation.

The present invention further encompasses methods and compositions forinhibiting or reducing activation of B cells, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a BLyS binding polypeptide in anamount effective to inhibit or reduce B cell activation.

The present invention further encompasses methods and compositions fordecreasing lifespan of B cells, comprising, or alternatively consistingof, contacting an effective amount of BLyS binding polypeptide withBLyS, wherein the effective amount of BLyS binding polypeptide inhibitsor reduces BLyS regulated lifespan of B cells.

The present invention further encompasses methods and compositions fordecreasing lifespan of B cells, comprising, or alternatively consistingof, administering to an animal in which such decrease is desired, a BLySbinding polypeptide in an amount effective to decrease B cell lifespan.

The present invention further encompasses methods and compositions forinhibiting or reducing graft rejection, comprising, or alternativelyconsisting of, administering to an animal in which such inhibition orreduction is desired, a BLyS binding polypeptide in an amount effectiveto inhibit or reduce graft rejection.

The present invention further encompasses methods and compositions forkilling cells of hematopoietic origin, comprising, or alternativelyconsisting of, contacting BLyS binding polypeptides with BLyS to form acomplex; and contacting the complex with cells of hematopoietic origin.

The present invention further encompasses methods and compositions forkilling cells of hematopoietic origin, comprising, or alternativelyconsisting of, administering to an animal in which such killing isdesired, a BLyS binding polypeptide in an amount effective to kill cellsof hematopoietic origin.

The present invention further encompasses methods and compositions forstimulating immunoglobulin production, comprising, or alternativelyconsisting of, contacting an effective amount of BLyS bindingpolypeptide with BLyS, wherein the effective amount of the BLyS bindingpolypeptide stimulates BLyS mediated immunoglobulin production.

The present invention further encompasses methods and compositions forstimulating immunoglobulin production comprising, or alternativelyconsisting of, administering to an animal in which such stimulation isdesired, a BLyS binding polypeptide in an amount effective to stimulateimmunoglobulin production.

The present invention further encompasses methods and compositions forstimulating B cell proliferation, comprising, or alternativelyconsisting of, contacting an effective amount of BLyS bindingpolypeptide with BLyS, wherein the effective amount of BLyS bindingpolypeptide stimulates BLyS mediated B cell proliferation.

The present invention further encompasses methods and compositions forstimulating B cell proliferation, comprising, or alternativelyconsisting of, administering to an animal in which such stimulation isdesired, a BLyS binding polypeptide in an amount effective to stimulateB cell proliferation.

The present invention further encompasses methods and compositions forincreasing activation of B cells, comprising, or alternativelyconsisting of, contacting an effective amount of BLyS bindingpolypeptide with BLyS, wherein the effective amount of BLyS bindingpolypeptide increases BLyS mediated activation of B cells.

The present invention further encompasses methods and compositions forincreasing activation of B cells, comprising, or alternativelyconsisting of, administering to an animal in which such increase isdesired, a BLyS binding polypeptide in an amount effective to increase Bcell activation.

The present invention further encompasses methods and compositions forincreasing lifespan of B cells, comprising, or alternatively consistingof, contacting an effective amount of BLyS binding polypeptide withBLyS, wherein the effective amount of BLyS binding polypeptide increasesBLyS regulated lifespan of B cells.

The present invention further encompasses methods and compositions forincreasing lifespan of B cells, comprising, or alternatively consistingof, administering to an animal in which such increase is desired, a BLySbinding polypeptide in an amount effective to increase lifespan of Bcells.

Definitions

In order that the invention may be clearly understood, the followingterms are defined:

The term “recombinant” is used to describe non-naturally altered ormanipulated nucleic acids, host cells transfected with exogenous nucleicacids, or polypeptide molecules that are expressed non-naturally,through manipulation of isolated nucleic acid (typically, DNA) andtransformation or transfection of host cells. “Recombinant” is a termthat specifically encompasses nucleic acid molecules that have beenconstructed in vitro using genetic engineering techniques, and use ofthe term “recombinant” as an adjective to describe a molecule,construct, vector, cell, polypeptide or polynucleotide specificallyexcludes naturally occurring such molecules, constructs, vectors, cells,polypeptides or polynucleotides.

The term “bacteriophage” is defined as a bacterial virus containing anucleic acid core and a protective shell built up by the aggregation ofa number of different protein molecules. The terms “bacteriophage” and“phage” are synonymous and are used herein interchangeably.

The term “affinity ligand” is sometimes used herein and is synonymouswith BLyS binding polypeptides.

The term “BLyS protein” as used herein encompasses both the membrane(e.g., SEQ ID NOs:173 and 174) and soluble forms (e.g., amino acids134-285 of SEQ ID NO:173) of BLyS. BLyS protein may be monomeric,dimeric, or trimeric or multivalent. Preferably, BLyS proteins arehomotrimeric.

The term “BLyS-like polypeptide” as used herein encompasses natural BLySor full-length recombinant BLyS as well as fragments and variantsthereof, such as, a modified or truncated form of natural BLyS orfull-length recombinant BLyS, which BLyS and BLyS-like polypeptideretain a BLyS functional activity. BLyS or BLyS fragments that may bespecifically bound by the compositions useful according to the inventioninclude, but are not limited to, human BLyS (SEQ ID NOs:173 and/or 174)or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOs:175 and/or176) or BLyS expressed on murine monocytes; rat BLyS (either the solubleforms as given in SEQ ID NOs:177, 178, 179 and/or 180 or in a membraneassociated form, e.g., on the surface of rat monocytes); or monkey BLyS(e.g., the monkey BLyS polypeptides of SEQ ID NOS:181 and/or 182, thesoluble form of monkey BLyS, or BLyS expressed on monkey monocytes) orfragments thereof. Preferably compositions useful according to theinvention bind human BLyS (SEQ ID NOs:173 and/or 174) or fragmentsthereof. BLyS and BLyS-like polypeptides retain at least one functionalactivity of the natural or full-length BLyS, including but not limitedto the following activities: binding to BLyS receptor (e.g., TACI(GenBank accession number AAC51790), and BCMA (GenBank accession numberNP_(—)001183)), stimulating B cell proliferation, stimulatingimmunoglobulin secretion by B cells, stimulating the BLyS receptorsignaling cascade and/or being bound by an anti-BLyS antibody or otherBLyS binding polypeptide. Assays that can be used to determine thefunctional activities of BLyS or BLyS like polypeptides can readily bedetermined by one skilled in the art (e.g., see assays disclosed inMoore et al., 1999, supra) “BLyS-like polypeptides” also include fusionpolypeptides in which all or a portion of BLyS is fused or conjugated toanother polypeptide. BLyS-like polypeptides that are fusion polypeptidesretain at least one functional activity of BLyS, preferably the abilityto stimulate B lymphocytes (see, for example, Moore et al., Science,285: 260-263 (1999)), to bind the BLyS receptors (e.g., TACI or BCMA),and/or to be bound by an anti-BLyS antibody or other BLyS bindingpolypeptide. BLyS fusion polypeptides may be made by recombinant DNAtechniques in which a gene or other polynucleotide coding sequence forBLyS or a fragment thereof is ligated in-frame (recombined) with thecoding sequence of another protein or polypeptide. The resultingrecombinant DNA molecule is then inserted into any of a variety ofplasmid or phage expression vectors, which enable expression of thefusion protein molecule in an appropriate eukaryotic or prokaryotic hostcell. BLyS fusion polypeptides may be generated by synthetic orsemi-synthetic procedures as well.

The terms “BLyS target” or “BLyS target protein” are sometimes usedherein and encompass BLyS and/or BLyS-like polypeptides. Thus, the BLySbinding polypeptides used according to the methods of the invention bind“BLyS target proteins” and can be used to bind, detect, remove, and/orpurify “BLyS target proteins.”

The term “binding polypeptide” is used herein to refer to anypolypeptide capable of forming a binding complex with another molecule,polypeptide, peptidomimetic or transformant.

A “BLyS binding polypeptide” is a molecule that can bind BLyS targetprotein. Non-limiting examples of BLyS binding polypeptides useful inthe methods of the invention are the polypeptide molecules having anamino acid sequence described herein (see SEQ ID NOs:1-12, 20-172, and186-444). The term BLyS binding polypeptide also encompasses BLySbinding fragments and variants (including derivatives) of polypeptideshaving the specific amino acid sequences described herein (SEQ IDNOs:1-12, 20-172, and 186-444). By “variant” of an amino acid sequenceas described herein is meant a polypeptide that binds BLyS, but does notnecessarily comprise an identical or similar amino acid sequence of aBLyS binding polypeptide specified herein. BLyS binding polypeptidesuseful according to the invention which are variants of a BLyS bindingpolypeptide specified herein satisfy at least one of the following: (a)a polypeptide comprising, or alternatively consisting of, an amino acidsequence that is at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% least99%, or 100% identical to the amino acid sequence of a BLyS bindingpolypeptide sequence disclosed herein (SEQ ID NOs:1-12, 20-172, and186-444), (b) a polypeptide encoded by a nucleotide sequence, thecomplementary sequence of which hybridizes under stringent conditions toa nucleotide sequence encoding a BLyS binding polypeptide disclosedherein (e.g., a nucleic acid sequence encoding the amino acid sequenceof SEQ ID NOs:1-12, 20-172, and 186-444), and/or a fragment of a BLySbinding polypeptide disclosed herein, of at least 5 amino acid residues,at least 10 amino acid residues, at least 15 amino acid residues, or atleast 20 amino acid residues. BLyS binding polypeptides useful accordingto the invention also encompass polypeptide sequences that have beenmodified for various applications provided that such modifications donot eliminate the ability to bind a BLyS target. Specific, non-limitingexamples of modifications contemplated include C-terminal or N-terminalamino acid substitutions or peptide chain elongations for the purpose oflinking the BLyS binder to a chromatographic material or other solidsupport. Other substitutions contemplated herein include substitution ofone or both of a pair of cysteine residues that normally form disulfidelinks, for example with non-naturally occurring amino acid residueshaving reactive side chains, for the purpose of forming a more stablebond between those amino acid positions than the former disulfide bond.All such modified binding polypeptides are also considered BLyS bindingpolypeptides so long as the modified polypeptides retain the ability tobind BLyS and/or BLyS-like polypeptides, and therefore, may be used inone or more of the various methods described herein, such as, to detect,purify, or isolate BLyS or BLyS-like polypeptides in or from a solution.BLyS binding polypeptides also include variants of the specific BLySbinding polypeptide sequences disclosed herein (e.g., SEQ ID NOs:1-12,20-172, and 186-444) which have an amino acid sequence corresponding toone of these polypeptide sequences, but in which the polypeptidesequence is altered by substitutions, additions or deletions thatprovide for molecules that bind BLyS. Thus, the BLyS bindingpolypeptides include polypeptides containing, as a primary amino acidsequence, all or part of the particular BLyS binding polypeptidesequence including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequence,resulting in a peptide which is functionally active. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Conservative substitutionsfor an amino acid within the sequence may be selected from other membersof the class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Such BLyS bindingpolypeptides can be made either by chemical peptide synthesis or byrecombinant production from a nucleic acid encoding the BLyS bindingpolypeptide which nucleic acid has been mutated. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson etal., J. Biol. Chem., 253:6551 (1978)), use of TAB.RTM. linkers(Pharmacia), etc.

As used and understood herein, percent homology or percent identity oftwo amino acid sequences or of two nucleic acid sequences is determinedusing the algorithm of Karlin and Atschul (Proc. Natl. Acad. Sci. USA,87: 2264-2268 (1990)), modified as in Karlin and Altschul (Proc. Natl.Acad. Sci. USA, 90: 5873-5877 (1993)). Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol.,215: 403-410 (1990)). BLAST nucleotide searches are performed with theNBLAST program to obtain nucleotide sequences homologous to a nucleicacid molecule described herein. BLAST protein searches are performedwith the XBLAST program to obtain amino acid sequences homologous to areference polypeptide. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul et al.(Nucleic Acids Res., 25: 3389-3402 (1997)). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used. See, http://www.ncbi.nlm.nih.gov.Alternatively, the percent identity of two amino acid sequences or oftwo nucleic acid sequences can be determined once the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino acid or nucleic acid sequence). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide at thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions×100%). In one embodiment, the twosequences are the same length.

The term “polypeptide”, as used herein, refers to a linear, branched, orcyclic (e.g., containing a loop structure) polymer of two or more aminoacid residues linked with a peptide bond. The term “polypeptide” is notrestricted to any particular upper limit of amino acid residues. Thus,the BLyS affinity ligands that comprise an amino acid sequence describedherein are properly referred to as “BLyS binding polypeptides” becausesuch binding polypeptides contain at least two amino acid residues heldtogether by a peptide bond, even though such molecules may also containone or more additional moieties or groups that are not amino acids, suchas N-terminal and/or C-terminal capping or functional groups, and thatmay or may not be involved in a peptide bond. The polypeptides may bemonovalent, divalent, trivalent, or multivalent and may comprise one ormore of the BLyS binding polypeptides having the amino acid sequence ofSEQ ID NOs:1-12, 20-172, and 186-444 and/or fragments or variantsthereof. The term “peptide” is used herein to have the same meaning as“polypeptide.”

The term “antibody,” as used herein, refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody fragments as well as variants(including derivatives) of antibodies and antibody fragments. Examplesof molecules which are described by the term “antibody” in thisapplication include, but are not limited to: single chain Fvs (scFvs),Fab fragments, Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs),Fvs, and fragments comprising or alternatively consisting of, either aVL or a VH domain. The term “single chain Fv” or “scFv” as used hereinrefers to a polypeptide comprising a VL domain of antibody linked to aVH domain of an antibody.

“Feed stream”: BLyS and BLyS-like polypeptides that are bound by a BLySbinding polypeptide of this invention may be produced by any methodknown in the art, including, but not limited to, chemical synthesis;production in transformed host cells; secretion into culture medium bynaturally occurring cells or recombinantly transformed bacteria, yeasts,fungi, insect cells, plant cells, and mammalian cells; production ingenetically engineered organisms (for example, transgenic mammals); andproduction in non-genetically engineered organisms. The solution,sample, or mixture that contains a BLyS or BLyS-like polypeptide as itis produced or is found present in a production solution will sometimesbe referred to as the “feed stream”.

The term “binding” refers to the determination by standard techniquesthat a binding polypeptide recognizes and binds to a given target. Suchstandard techniques include, but are not limited to, affinitychromatography, equilibrium dialysis, gel filtration, enzyme linkedimmunosorbent assay (ELISA), FACS analysis, and the monitoring ofspectroscopic changes that result from binding, e.g., using fluorescenceanisotropy, either by direct binding measurements or competition assayswith another binder.

The term “specificity” refers to a binding polypeptide useful accordingto the invention that has a higher binding affinity for one target overanother. Thus, the term “BLyS target protein specificity” refers to amolecule having a higher affinity for BLyS target protein as comparedwith another molecule that is not a BLyS target protein.

The term “epitopes” as used herein refers to portions of BLyS havingantigenic or immunogenic activity in an animal, preferably a mammal. Anepitope having immunogenic activity is a portion of BLyS that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of BLyS to which an antibody or BLyS binding polypeptidespecifically binds as determined by any method known in the art, forexample, by the immunoassays described herein. Antigenic epitopes neednot necessarily be immunogenic.

The term “fragment” as used herein refers to a polypeptide comprising anamino acid sequence of at least 5 amino acid residues, at least 6 aminoacid residues, at least 7 amino acid residues, at least 8 amino acidresidues, at least 9 amino acid residues, at least 10 amino acidresidues, at least 11 amino acid residues, at least 12 amino acidresidues, at least 13 amino acid residues, at least 14 amino acidresidues, at least 15 amino acid residues, at least 16 amino acidresidues, at least 17 amino acid residues, at least 18 amino acidresidues, at least 19 amino acid residues, at least 20 amino acidresidues, at least 21 amino acid residues, at least 22 amino acidresidues, at least 23 amino acid residues, at least 24 amino acidresidues, or at least 25 amino acid residues of the amino acid sequenceof BLyS, or a BLyS binding polypeptide (including molecules thatcomprise, or alternatively consist of, BLyS binding polypeptidefragments or variants thereof).

The term “fusion protein” as used herein refers to a polypeptide thatcomprises, or alternatively consists of, an amino acid sequence of aBLyS binding polypeptide and an amino acid sequence of a heterologouspolypeptide (i.e., a polypeptide unrelated to the BLyS bindingpolypeptide).

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny may not be identical to theparent cell transfected with the nucleic acid molecule due to mutationsor environmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

Other terms are defined as necessary in the text below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for detecting,diagnosing, prognosing, and/or monitoring diseases or disordersassociated with aberrant BLyS or BLyS receptor expression orinappropriate BLyS or BLyS receptor function in an animal, preferably amammal, and most preferably a human, comprising, or alternativelyconsisting of, use of BLyS binding polypeptides (including moleculeswhich comprise, or alternatively consist of, BLyS binding polypeptidefragments or variants thereof) that specifically bind to BLyS. Diseasesand disorders which can be detected, diagnosed, prognosed and/ormonitored with the BLyS binding polypeptides include, but are notlimited to, immune system diseases or disorders (e.g., autoimmunediseases or disorders, immunodeficiencies, lupus, glomerular nephritis,rheumatoid arthritis, multiple sclerosis, graft vs. host disease,myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome),proliferative diseases or disorders (e.g., cancer, premalignantconditions, benign tumors, hyperproliferative disorders, benignproliferative disorders, leukemia, lymphoma, chronic lymphocyticleukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, Tcell proliferative diseases and disorders, B cell proliferative diseasesand disorders, monocytic proliferative diseases or disorders, acutemyelogenous leukemia, macrophage proliferative diseases and disorders,and carcinoma), infectious diseases (e.g., AIDS), and inflammatorydisorders (e.g., asthma, allergic disorders, and rheumatoid arthritis).

The present invention further encompasses methods and compositions forpreventing, treating and/or ameliorating diseases or disordersassociated with aberrant BLyS or BLyS receptor expression orinappropriate BLyS or BLyS receptor function in an animal, preferably amammal, and most preferably a human, comprising, or alternativelyconsisting of, administering to an animal in which such treatment,prevention or amelioration is desired one or more BLyS bindingpolypeptides (including molecules which comprise, or alternativelyconsist of, BLyS binding polypeptide fragments or variants thereof) inan amount effective to treat, prevent or ameliorate the disease ordisorder. Diseases and disorders which can be prevented, treated, and/orameliorated with the BLyS binding polypeptides include, but are notlimited to, immune system diseases or disorders (e.g., autoimmunediseases or disorders, immunodeficiencies, lupus, glomerular nephritis,rheumatoid arthritis, multiple sclerosis, graft vs. host disease,myasthenia gravis, Hashimoto's disease, immunodeficiency syndrome,hypogammaglobulinemia, and hypergammaglobulinemia), proliferativediseases or disorders (e.g., cancer, premalignant conditions, benigntumors, hyperproliferative disorders, benign proliferative disorders,leukemia, lymphoma, chronic lymphocytic leukemia, multiple myeloma,Hodgkin's lymphoma, Hodgkin's disease, T cell proliferative diseases anddisorders, B cell proliferative diseases and disorders, monocyticproliferative diseases or disorders, acute myelogenous leukemia,macrophage proliferative diseases and disorders, and carcinoma),infectious diseases (e.g., AIDS), and inflammatory disorders (e.g.,asthma, allergic disorders, and rheumatoid arthritis).

BLyS Binding Polypeptides

The methods of the present invention may be performed utilizing newpolypeptides and families of polypeptides that specifically bind to Blymphocyte stimulator protein (BLyS) and/or BLyS-like polypeptides. Inparticular, the invention encompasses diagnostic and therapeutic usesfor polypeptides that specifically bind to a polypeptide or polypeptidefragment of human BLyS (SEQ ID NOs:173 and/or 174) or BLyS expressed onhuman monocytes; murine BLyS (SEQ ID NOs:175 and/or 176) or BLySexpressed on murine monocytes; rat BLyS (either the soluble forms asgiven in SEQ ID NOs:177, 178, 179 and/or 180 or in a membrane associatedform, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., themonkey BLyS polypeptides of SEQ ID NOS:181 and/or 182, the soluble formof monkey BLyS, or BLyS expressed on monkey monocytes); preferably humanBLyS.

In preferred embodiments, the BLyS binding polypeptides used accordingto the present invention (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof), specifically bind to BLyS and do not cross-react withany other antigens. In more preferred embodiments, the BLyS bindingpolypeptides specifically bind to BLyS and do not cross-react with TRAIL(Hahne et al., J. Exp. Med., 188(6):1185-90 (1998)), APRIL (Wilet etal., Immunity, 3(6):673-82 (1995)), Endokine-alpha (Kwon et al., J.Biol. Chem., 274(10):6056-61 (1999)), TNF-alpha, TNF-beta (Nedwin etal., J. Immunol., 135(4):2492-7 (1985)), Fas-L (Suda et al., Cell,75(6):1169-78 (1993)), or LIGHT (Mauri et al., Immunity, 8(1):21-30(1998)).

Many BLyS binding polypeptides have been discovered which may be used inthe methods of the present invention. Specific BLyS binding polypeptidesfor use in the present invention comprise, or alternatively consist of,an amino acid sequence selected from the group consisting of SEQ IDNOs:1-12, 20-172, and 186-444, preferably SEQ ID NOs:163-172 or 436-444as referred to above and in Tables 1-8, 13 and 14. In its broadestaspects, the methods of the present invention may be carried out using apolypeptide capable of binding to BLyS and comprising the polypeptideAsp-Xaa-Leu-Thr (SEQ ID NO:446), where Xaa is Pro, Ser, Thr, Phe, Leu,Tyr, Cys, or Ala (preferably Pro or Ser).

Additional polypeptides for use in the methods described herein includepolypeptides with the potential to form a cyclic or loop structurebetween invariant Cys residues comprising, or alternatively consistingof, an amino acid sequence selected from A-E (SEQ ID NOs:1-5): (A)X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃, (SEQ ID NO:1)whereinX₁ is Ala, Asn, Lys, or Ser;X₂ is Ala, Glu, Met, Ser, or Val;X₃ is Ala, Asn, Lys, or Pro (preferably Lys);X₅ is Phe, Trp, or Tyr (preferably Tyr);X₇ is Pro or Tyr (preferably Pro);X₁₁ is Ala, Gln, His, Phe, or Val;X₁₂ is Asn, Gln, Gly, His, Ser, or Val; andX₁₃ is Ala, Asn, Gly, Ile, Pro, or Ser,

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(B) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄, (SEQ ID NO:2)whereinX₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, Val, or is absent;X₂ is Ala, Asn, Asp, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val;X₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Trp, Tyr, or Val (preferably Asp);X₅ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val(preferably Glu or Leu);X₇ is His, Leu, Lys, or Phe (preferably His or Leu);X₈ is Leu, Pro, or Thr (preferably Thr or Pro);X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys);X₁₀ is Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr, orVal;X₁₂ is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val;X₁₃ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val; andX₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro,Trp, Tyr, Val, or is absent,

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(C) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅, (SEQ ID NO:3)whereinX₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr;X₂ is Asn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp;X₃ is Ala, Arg, Asn, Gln, Glu, His, Phe, Pro, or Thr (preferably Ala);X₅ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);X₇ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);X₉ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);X₁₀ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;X₁₁ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser);X₁₃ is Gln, Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val (preferably Val);X₁₄ is Asn, Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; andX₁₅ is Asn, Asp, Glu, Leu, Lys, Met, Pro, or Thr (preferably Glu orPro),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(D) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆, (SEQ IDNO:4)whereinX₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent (preferablySer);X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferably Arg);X₃ is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp);X₅ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;X₆ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;X₇ is Asp, His, Leu, or Ser (preferably Asp);X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);X₉ is Ala, Arg, Asn, or Leu (preferably Leu);X₁₀ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);X₁₁ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val;X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu);X₁₅ is Asn, His, Leu, Pro, or Tyr (preferably His, Leu or Pro); andX₁₆ is Asn, Asp, His, Phe, Ser, or Tyr, (preferably Asp or Ser),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(E) X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈,(SEQ ID NO:5)whereinX₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or is absent(preferably Arg);X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or is absent (preferably Asn,Asp, Gly, or Pro);X₃ is Arg, Asn, Gln, Glu, Gly, Lys, Met, Pro, Trp or Val (preferably Glyor Met);X₅ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val(preferably Trp, Tyr, or Val);X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferablyAsp);X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);X₉ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr(preferably Lys or Thr);X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferablyArg or Leu);X₁₂ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr(preferably Thr or Trp);X₁₃ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr(preferably Met or Phe);X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, orVal (preferably Val);X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro, Ser, or Trp (preferablyMet);X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr, (preferablyArg, His, or Tyr); andX₁₈ is Ala, Arg, Asn, Asp, His, Leu, Phe, or Trp (preferably His orAsn),wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

Additional BLyS binding polypeptides that may be used in the methods ofthe present invention include linear polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from F andG (SEQ ID NOs:6 and 7): (F) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂, (SEQID NO:6)whereinX₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val(preferably Gly, Tyr, or Val);X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe, Thr, Trp, or Tyr (preferablyHis or Tyr);X₃ is Ala, Asp, Lys, Phe, Thr, Trp or Tyr (preferably Asp or Tyr);X₄ is Arg, Asp, Gln, Lys, Met, Phe, Pro, Ser, Tyr, or Val (preferablyAsp or Gln);X₅ is Asp, Leu, Lys, Phe, Pro, Ser, or Val (preferably Leu or Ser);X₆ is His, Ile, Leu, Pro, Ser, or Thr (preferably Leu or Thr);X₇ is Arg, Gly, His, Leu, Lys, Met, or Thr (preferably Lys or Thr);X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, or Thr (preferably Leu or Lys);X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His, Leu, Met, Ser, Trp, Tyr, or Val(preferably Met or Ser);X₁₀ is Ile, Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably Thr orLeu);X₁₁ is Ala, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, orVal (preferably Pro or Thr); andX₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val(preferably Arg or Pro),

wherein said polypeptide binds BLyS and/or BLyS-like polypeptides; or(G) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃, (SEQ ID NO:7)whereinX₁ is Asp, Gln, Glu, Gly, His, Lys, Met, or Trp (preferably Glu, Lys);X₂ is Arg, Gln, His, Ile, Leu, or Pro (preferably His or Pro);X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably Tyr);X₄ is Asn, Asp, Gln, Glu, Met, Pro, Ser, or Tyr (preferably Asp or Gln);X₅ is Asn, Asp, His, Ile, Leu, Met, Pro, Thr or Val (preferably Asn orThr);X₆ is Asp, Glu, His, Leu, Lys, Pro, or Val (preferably Asp or Pro);X₇ is Arg, Asn, Gln, His, Ile, Leu, Met, Pro, or Thr (preferably Ile orPro);X₈ is Gln, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or Thr);X₉ is Asn, Gln, Gly, His, Leu, Lys, Ser, or Thr (preferably Lys);X₁₀ is Ala, Gly, Ile, Leu, Lys, Met, or Phe (preferably Gly or Met);X₁₁ is Ala, Glu, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val(preferably Ala or Thr);X₁₂ is Arg, Gln, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg orHis); andX₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val (preferably His),wherein said polypeptide binds BLyS and/or BLyS-like polypeptides.

Additional BLyS binding polypeptides that may be used in the methods ofthe present invention include BLyS binding polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from H-L(SEQ ID NOs:8-12): (H) Cys-X₂-Phe-X₄-Trp-Glu-Cys, (SEQ ID NO:8)whereinX₂ is Phe, Trp, or Tyr (preferably Tyr); and

X₄ is Pro or Tyr (preferably Pro); or (I) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys,(SEQ ID NO:9)whereinX₂ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu);X₃ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val(preferably Glu or Leu);X₄ is His, Leu, Lys, or Phe (preferably His or Leu);X₅ is Leu, Pro, or Thr (preferably Thr or Pro);X₆ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys); and

X₇ is Ala, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Trp, Tyr,or Val; or (J) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys, (SEQ ID NO:10)whereinX₂ is Asn, Asp, Pro, Ser, or Thr (preferably Asp);X₃ is Arg, Asp, Ile, Leu, Met, Pro, or Val (preferably Ile);X₄ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Val or Leu);X₅ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr);X₆ is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu);X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp;

X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); or (K)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys, (SEQ ID NO:11)whereinX₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr;X₃ is His, Ile, Leu, Met, Phe, Pro, Trp, or Tyr;X₄ is Asp, His, Leu, or Ser (preferably Asp);X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu or Pro);X₆ is Ala, Arg, Asn, or Leu (preferably Leu);X₇ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr);X₈ is Ala, Arg, Asn, Gly, His, Lys, Ser, or Tyr;

X₉ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, or Val; or (L)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys, (SEQ ID NO:12)whereinX₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val(preferably Trp, Tyr, or Val);X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr (preferablyAsp);X₄ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr (preferably Asp);X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr (preferably Leu);X₆ is Asp, Leu, Pro, Thr, or Val (preferably Leu or Thr);X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr(preferably Lys or Thr);X₈ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, or Thr (preferablyArg or Leu);X₉ is Ala, Asn, Gln, Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr(preferably Thr or Trp);X₁₀ is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr(preferably Met or Phe);X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr, orVal (preferably Val);wherein said polypeptides bind BLyS and/or BLyS-like polypeptides.

Additional BLyS binding polypeptides that may be used in the methods ofthe present invention include linear polypeptides comprise the followingamino acid sequence M (SEQ ID NO:447): (M)Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄, (SEQ ID NO:447)whereinX₂ is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu, Met, Thr,Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid except Arg;preferably Asn);X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);X₄ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);X₆ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);X₉ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);X₁₁ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp);X₁₂ is Leu, Phe, Val, Ile, or His (preferably Leu);X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr, Val, Ala, Cys, Ile, Phe,or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, or Trp; preferably Pro); andX₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly, Ser, Tyr, Ala, Cys, Lys,Ile, Thr or Leu (i.e., not Phe, Met, Pro, or Trp; preferably Asp, Val orGlu).

Preferred BLyS binding polypeptides that may be used in the methods ofthe present invention include linear polypeptides comprising a coresequence of the formula N: (N) X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀, (SEQID NO:448)whereinX₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferably Trp);X₂ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr);X₄ is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser);X₇ is Lys, Asn, Gln, Gly, or Arg (preferably Lys);X₉ is Trp, Ser, Thr, Arg, Cys, Tyr, or Lys (preferably Trp); andX₁₀ is Leu, Phe, Val, Ile, or His (preferably Leu).

Especially preferred BLyS binding polypeptides that may be used in themethods of the present invention include linear polypeptides comprisingthe core peptide Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ IDNO:436).

In performing certain methods according to the present invention, it ispreferred that the BLyS binding polypeptides, or phage displaying suchbinding polypeptides, irreversibly bind the BLyS protein in its native,soluble trimeric form.

In performing certain methods according to the present invention, it ispreferred that the BLyS binding polypeptides of the present invention,or phage displaying such binding polypeptides, reversibly bind the BLySprotein in its native, soluble trimeric form.

In performing certain methods according to the invention, it may beadvantageous for a BLyS binding polypeptide to bind BLyS target proteinwith high affinity. In specific embodiments, BLyS binding polypeptidesused in this invention will bind BLyS target proteins with adissociation constant or K_(D) of less than or equal to 5×10⁻² M, 10⁻²M, 5×10⁻³ M, 10⁻³ M, 5×10 ⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. Morepreferably, BLyS binding polypeptides used in the invention will bindBLyS target proteins with a dissociation constant or K_(D) less than orequal to 5×10 ⁻⁶ M, 10⁻⁶ M, 5×10 ⁻⁷ M, 10⁻⁷M, 5×10 ⁻⁸ M, or 10⁻⁸ M. Evenmore preferably, BLyS binding polypeptides used in the methods of theinvention bind BLyS target proteins with a dissociation constant orK_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M, 5×10 ⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10 ⁻¹³ M, 10⁻¹³ M, 5×10 ⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In certain preferred embodiments, BLyS binding polypeptides reversiblybind BLyS and/or BLyS-like polypeptides and release bound BLyS proteinin an active form, preferably in the native soluble trimeric form, underspecific release conditions. In specific embodiments, BLyS bindingpolypeptides bind BLyS target proteins with off-rates or k_(off) greaterthan or equal to 10⁻¹⁰ s⁻¹, 5×10 ⁻⁹ s⁻¹, 10⁻⁹ s⁻¹, 5×10⁻⁸ s⁻¹, 10⁻⁸ s⁻¹,5×10⁻⁷ s⁻¹, 10⁻⁷ s⁻¹, 5×10⁻⁶ s⁻¹, 10⁻⁶ s⁻¹, 5×10⁻⁵ s⁻¹, 10⁻⁻⁵ s⁻¹,5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻³ s⁻¹, 10⁻³ s⁻¹, 5×10⁻² s⁻¹, 10⁻² s⁻¹, 5×10⁻¹, or 10⁻¹ s⁻¹.

Binding experiments to determine K_(D) and off-rates can be performed ina number of conditions including, but not limited to, [pH 6.0, 0.01%Tween 20], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween 20], [pH9.0, 0.1%Tween 20], [pH6.0, 15% ethylene glycol, 0.01% Tween 20], [pH5.0, 15%ethylene glycol, 0.01% Tween 20], and [pH9.0, 15% ethylene glycol, 0.01%Tween 20] The buffers in which to make these solutions can readily bedetermined by one of skill in the art, and depend largely on the desiredpH of the final solution. Low pH solutions (<pH 5.5) can be made, forexample, in citrate buffer, glycine-HCl buffer, or in succinic acidbuffer. High pH solutions can be made, for example, in Tris-HCl,phosphate buffers, or sodium bicarbonate buffers. A number of conditionsmay be used to determine K_(D) and off-rates for the purpose ofdetermining, for example, optimal pH and/or salt concentrations.

In certain embodiments, BLyS binding polypeptides reversibly bind BLySand/or BLyS-like polypeptides, preferably in the native soluble,trimeric form.

In preferred embodiments, BLyS binding polypeptides reversibly bind onlythe native soluble, trimeric form of BLyS.

In certain embodiments, BLyS binding polypeptides irreversibly bind BLySand/or BLyS-like polypeptides, preferably in the native soluble,trimeric form.

In preferred embodiments, BLyS binding polypeptides irreversibly bindonly the native soluble, trimeric form of BLyS.

In some screening or assay procedures, it is possible and moreconvenient to use recombinant bacteriophage that display a particularBLyS binding polypeptide instead of using isolated BLyS bindingpolypeptide. Such procedures include phage-based ELISA protocols andimmobilization of phage displaying a binding polypeptide tochromatographic materials. Such screening assays and procedures areroutine in the art and may be readily adapted for procedures usingrecombinant bacteriophage such as disclosed herein.

Specific methods of the present invention contemplate the use of BLySbinding polypeptides that competitively inhibit the binding of a BLySbinding molecule. Competitive inhibition can be determined by anysuitable method known in the art, for example, using the competitivebinding assays described herein. In preferred embodiments, thepolypeptide competitively inhibits the binding of a BLyS bindingmolecule to BLyS by at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 60%, or at least 50%. In amore preferred embodiment, the BLyS binding polypeptide competitivelyinhibits the binding of a BLyS binding molecule to the native solubletrimeric form of BLyS, by at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may have one or more of the same biological characteristics asone or more of the BLyS binding polypeptides specifically describedherein. By “biological characteristics” is meant, the in vitro or invivo activities or properties of the BLyS binding polypeptides, such as,for example, the ability to bind to BLyS (e.g., the soluble form ofBLyS, the membrane-bound form of BLyS, the soluble form andmembrane-bound form of BLyS), and/or an antigenic and/or epitope regionof BLyS), the ability to substantially block BLyS/BLyS receptor (e.g.,TACI and BCMA) binding, the ability to substantially increase BLyS/BLySreceptor (e.g., TACI and BCMA) binding, the ability to block BLySmediated biological activity (e.g., stimulation of B cell proliferationand immunoglobulin production), or, the ability to enhance or stimulateBLyS mediated biological activity (e.g., stimulation of B cellproliferation and immunoglobulin production). Optionally, the BLySbinding polypeptides useful according to the invention will bind to thesame epitope as at least one of the BLyS binding polypeptidesspecifically referred to herein. Such epitope binding can be routinelydetermined using assays known in the art.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may be polypeptides that neutralize BLyS or a fragmentthereof. By a BLyS binding polypeptide that “neutralizes BLyS or afragment thereof” is meant a BLyS binding polypeptide that inhibits(i.e., is effective to reduce or abolish) or abolishes the ability ofBLyS: to bind to its receptor (e.g., TACI and BCMA), to stimulate B cellactivation, to stimulate B cell proliferation, to stimulateimmunoglobulin secretion by B cells, to increase B cell lifespan, and/orto stimulate the BLyS receptor signalling cascade.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may also be effective to inhibit or abolish BLyS-mediated Bcell proliferation as determined by any method known in the art such as,for example, the assays described in the Examples, infra, said BLySbinding polypeptides comprising, or alternatively consisting of, apolypeptide having an amino acid sequence of any one of SEQ ID NOs:1-12,20-172, and 186-444, preferably of SEQ ID NOs:163-172 and 436-444, or afragment or variant thereof.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may also be effective to enhance the activity of BLyS or afragment thereof, said BLyS binding polypeptides comprising, oralternatively consisting of, a polypeptide having an amino acid sequenceof any one of SEQ ID NOs:1-12, 20-172, and 186-444, preferably of SEQ IDNOs:163-172 or 436-444, or a fragment or variant thereof. By a BLySbinding polypeptide that “enhances the activity of BLyS or a fragmentthereof” is meant a BLyS binding polypeptide that increases the abilityof BLyS: to bind to its receptor (e.g., TACI and BCMA), to stimulate Bcell proliferation, to stimulate immunoglobulin secretion by B cells, toactivate B cells, to increase B cell lifespan and/or to stimulate a BLySreceptor signalling cascade (e.g., to activate calcium-modulator andcyclophilin ligand (“CAML”), calcineurin, nuclear factor of activated Tcells transcription factor (“NF-AT”), nuclear factor-kappa B (“NF-kappaB”), activator protein-1 (AP-1), SRF, extracellular-signal regulatedkinase 1 (ERK-1), polo like kinases (PLK), ELF-1, high mobility group I(HMG-I), and/or high mobility group Y (HMG-Y)). Nucleic acid moleculesencoding these BLyS binding polypeptides are also encompassed by theinvention.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may also be effective to stimulate BLyS mediated B cellproliferation as determined by any method known in the art, such as, forexample, the assays described in the Examples, infra, said BLyS bindingpolypeptides comprising, or alternatively consisting of, a polypeptidehaving an amino acid sequence of any one of SEQ ID NOs:1-12, 20-172, and186-444, preferably of SEQ ID NOs:163-172 or 436-444, or a fragment orvariant thereof. Nucleic acid molecules encoding these BLyS bindingpolypeptides are also encompassed by the invention.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) useful in the practice of the methods of the presentinvention may include polypeptides effective to specifically bind to thesoluble form of BLyS, polypeptides that specifically bind to themembrane-bound form of BLyS, and polypeptides that specifically bind toboth the soluble form and membrane-bound form of BLyS.

The methods of the present invention may also be carried out usingmixtures of BLyS binding polypeptides (including molecules comprising,or alternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) that specifically bind to BLyS, wherein the mixturecontains at least one, two, three, four, five or more different BLySbinding polypeptides. In particular, the invention provides for the useof mixtures of different BLyS binding polypeptides that specificallybind to the soluble form of BLyS, the membrane-bound form of BLyS,and/or both the membrane-bound form and soluble form of BLyS. Inspecific embodiments, the methods of the invention utilize mixtures ofat least 2, preferably at least 4, at least 6, at least 8, at least 10,at least 12, at least 15, at least 20, or at least 25 different BLySbinding polypeptides that specifically bind to BLyS, wherein at least 1,at least 2, at least 4, at least 6, or at least 10, BLyS bindingpolypeptides of the mixture are BLyS binding polypeptides.

The methods of the present invention may also be carried out usingpanels of BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) that specifically bind to BLyS, wherein the panel hasat least one, two, three, four, five or more different BLyS bindingpolypeptides. In particular, the invention provides for the use ofpanels of different BLyS binding polypeptides that specifically bind tothe soluble form of BLyS, the membrane-bound form of BLyS, and/or boththe membrane-bound form and soluble form of BLyS. In specificembodiments, the invention provides for the use of panels of BLySbinding polypeptides that have different affinities for BLyS, differentspecificities for BLyS, or different dissociation rates. The inventionprovides for the use of panels of at least 10, preferably at least 25,at least 50, at least 75, or at least 100 BLyS binding polypeptides.Panels of BLyS binding polypeptides can be used, for example, in 96 wellplates for assays such as ELISAs.

The methods of the present invention may also be carried out usingcompositions comprising one or more BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of BLyS bindingpolypeptide fragments or variants). In one embodiment, a compositionused in a method of the present invention comprises, one, two, three,four, five, or more BLyS binding polypeptides that comprise oralternatively consist of, a polypeptide having an amino acid sequence ofany one or more of the BLyS binding polypeptides contained in SEQ IDNOs:1-12, 20-172, and 186-444 as disclosed in Tables 1-8 and 13, or avariant thereof.

As discussed in more detail below, a composition useful in the methodsof the invention may be used either alone or in combination with othercompositions. The BLyS binding polypeptides (including moleculescomprising, or alternatively consisting of BLyS binding polypeptidefragments or variants of the present invention) may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, BLyS binding polypeptides of the present invention may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, polypeptide linkers, drugs, radionuclides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 0 396 387.

Production and Modification of BLyS Binding Polypeptides

BLyS binding polypeptides useful in practicing the methods of thepresent invention may be produced by chemical synthesis, semi-syntheticmethods, and recombinant DNA methodologies known in the art.

In certain embodiments, BLyS binding polypeptides of the presentinvention are produced by chemical or semi-synthetic methodologies knownin the art (see, Kelley et al. in Genetic Engineering Principles andMethods, Setlow, J. K., ed. (Plenum Press, NY., 1990), vol. 12, pp.1-19; Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,San Francisco, 1989). One advantage of these methodologies is that theyallow for the incorporation of non-natural amino acid residues into thesequence of the BLyS binding polypeptide.

In preferred embodiments, BLyS binding polypeptides are chemicallysynthesized (see, e.g., Merrifield, J. Am. Chem. Soc., 85: 2149 (1963);Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132 (1985)). For example,polypeptides can be synthesized by solid phase techniques, cleaved fromthe resin, and purified by preparative high performance liquidchromatography (see, e.g., Creighton, Proteins: Structures and MolecularProperties (W.H. Freeman and Co., N.Y., 1983), pp. 50-60). BLyS bindingpolypeptides can also be synthesized by use of a peptide synthesizer.The composition of the synthetic polypeptides may be confirmed by aminoacid analysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Co., N.Y., 1983), pp. 34-49). Furthermore, if desired, BLyS bindingpolypeptides may contain non-classical amino acids or chemical aminoacid analogs, which can routinely be introduced during chemicalsynthesis as a substitution or addition into the BLyS bindingpolypeptides. Non-classical amino acids include, but are not-limited to,the D-isomers of the common amino acids, 2,4-diaminobutyric acid,alpha-aminoisobutyric acid, 4-aminobutyric acid (4Abu), 2-aminobutyricacid (Abu), 6-aminohexanoic acid (epsilon-Ahx), 2-aminoisobutyric acid(Aib), 3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine (bAla), fluoro-amino acids, designer amino acids such asbeta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

Solid phase peptide synthesis begins at the carboxy (C) terminus of theputative polypeptide by coupling a protected amino acid to a suitableresin, which reacts with the carboxyl group of the C-terminal amino acidto form a bond that is readily cleaved later, for example, a halomethylresin such as chloromethyl resin, bromomethyl resin, hydroxymethylresin, aminomethyl resin, benzhydrylamine resin, ort-alkyloxycarbonyl-hydrazide resin. After removal of the α-aminoprotecting group with, for example, trifluoroacetic acid (TFA) inmethylene chloride and neutralization with, for example TEA, the nextcycle in the synthesis is ready to proceed. The remaining α-amino and,if necessary, side-chain-protected amino acids are then coupledsequentially in the desired order by condensation to obtain anintermediate compound connected to the resin. Alternatively, some aminoacids may be coupled to one another forming an oligopeptide prior toaddition to the growing solid phase polypeptide chain.

The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according tocondensation methods known in the art, including but not limited to, theazide method, mixed acid anhydride method, DCC(dicyclohexylcarbodiimide) method, active ester method (p-nitrophenylester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino)phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid imidoester method), and Woodward reagent K method.

Common to chemical synthesis of peptides is the protection or capping(blocking) of the reactive side chain groups of the various amino acidresidues with suitable protecting or capping groups at that site untilthe group is ultimately removed after the polypeptide chain has beencompletely assembled. Also common is the protection or capping of theα-amino group on an amino acid or a fragment while that entity reacts atthe carboxyl group followed by the selective removal of theα-amino-protecting group to allow subsequent reaction to take place atthat location. Accordingly, during synthesis, intermediate compounds areproduced which includes each of the amino acid residues located in thedesired sequence in the peptide chain with various of these residueshaving side-chain protecting or capping groups. These protecting orcapping groups on amino acid side chains are then removed substantiallyat the same time so as to produce the desired resultant productfollowing purification.

The typical protective, capping, or blocking groups for α- and ε-aminoside chain groups found in amino acids are exemplified bybenzyloxycarbonyl (Z), isonicotinyloxycarbonyl (iNOC),O-chlorobenzyloxycarbonyl [Z(NO₂)], p-methoxybenzyloxycarbonyl [Z(OMe)],t-butoxycarbonyl (Boc), t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl,adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc),9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyiethoxycarbonyl (Msc),trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS),diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl (Mpt), and thelike.

Protective, capping, or blocking groups for the carboxyl group of aminoacids include, for example, benzyl ester (OBzl), cyclohexyl ester (Chx),4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester(OPic), and the like. It is usually also desirable that side chaingroups of specific amino acids such as arginine, cysteine, and serine,are protected by a suitable protective group as occasion demands. Forexample, the guanidino group in arginine may be protected with nitro,p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group incysteine may be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethy-benzyl(Tmb), etc., and the hydroxyl group in the serine can be protected withbenzyl, t-butyl, acetyl, tetrahydropyranyl, etc.

After the desired amino acid sequence has been completed, theintermediate polypeptide is removed from the resin support by treatmentwith a reagent, such as liquid HF and one or more thio-containingscavengers, which cleaves the peptide molecule from the resin and allthe remaining side-chain protecting groups. Following HF cleavage, theprotein sequence is washed with ether, transferred to a large volume ofdilute acetic acid, and stirred at pH adjusted to about 8.0 withammonium hydroxide. Upon pH adjustment, the polypeptide takes itsdesired conformational arrangement.

By way of example but not by way of limitation, polypeptides can bechemically synthesized and purified as follows: Peptides can besynthesized by employing the N-alpha-9-fluorenylmethyloxycarbonyl orFmoc solid phase peptide synthesis chemistry using a Rainin SymphonyMultiplex Peptide Synthesizer. The standard cycle used for coupling ofan amino acid to the peptide-resin growing chain generally includes: (1)washing the peptide-resin three times for 30 seconds withN,N-dimethylformamide (DMF); (2) removing the Fmoc protective group onthe amino terminus by deprotection to with 20% piperdine in DMF by twowashes for 15 minutes each, during which process mixing is effected bybubbling nitrogen through the reaction vessel for one second every 10seconds to prevent peptide-resin settling; (3) washing the peptide-resinthree times for 30 seconds with DMF; (4) coupling the amino acid to thepeptide resin by addition of equal volumes of a 250 mM solution of theFmoc derivative of the appropriate amino acid and an activator mixconsisting or 400 mM N-methylmorpholine and 250 mM(2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6)washing the peptide-resin three times for 30 seconds of DMF. This cyclecan be repeated as necessary with the appropriate amino acids insequence to produce the desired peptide. Exceptions to this cycleprogram are amino acid couplings predicted to be difficult by nature oftheir hydrophobicity or predicted inclusion within a helical formationduring synthesis. For these situations, the above cycle can be modifiedby repeating step 4 a second time immediately upon completion of thefirst 45 minute coupling step to “double couple” the amino acid ofinterest. Additionally, in the first coupling step in peptide synthesis,the resin can be allowed to swell for more efficient coupling byincreasing the time of mixing in the initial DMF washes to three 15minute washes rather than three 30 second washes.

After peptide synthesis, the peptide can be cleaved from the resin asfollows: (1) washing the peptide-resin three times for 30 seconds withDMF; (2) removing the Fmoc protective group on the amino terminus bywashing two times for 15 minutes it 20% piperdine in DMF; (3) washingthe peptide-resin three times for 30 seconds with DMF; and (4) mixing acleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4%water, 2.4% phenol, and 0.2% triisopropysilane with the peptide-resinfor two hours, then filtering the peptide in the cleavage cocktail awayfrom the resin, and precipitating the peptide out of solution byaddition of two volumes of ethyl ether. Specifically, to isolate thepeptide, the ether-peptide solution can be allowed to sit at −20° C. for20 minutes, then centrifuged at 6,000×G for 5 minutes to pellet thepeptide, and the peptide can be washed three times with ethyl ether toremove residual cleavage cocktail ingredients. The final peptide productcan be purified by reversed phase high pressure liquid chromatography(RP-HPLC) with the primary solvent consisting of 0.1% TFA and theeluting buffer consisting of 80% acetonitrile and 0.1% TFA. The purifiedpeptide can then be lyophilized to a powder.

In other specific embodiments, branched versions of the BLyS bindingpolypeptides described herein are provided, e.g., by substituting one ormore amino acids within the BLyS binding polypeptide sequence with anamino acid or amino acid analog with a free side chain capable offorming a peptide bond with one or more amino acids (and thus capable offorming a “branch”).

Branched peptides may be prepared by any method known in the art forcovalently linking any naturally occurring or synthetic amino acid toany naturally occurring or synthetic amino acid in a peptide chain whichhas a side chain group able to react with the amino or carboxyl group onthe amino acids so as to become covalently attached to the peptidechain. In particular, amino acids with a free amino side chain group,such as, but not limited to, diaminobutyric acid, lysine, arginine,ornithine, diaminopropionic acid and citrulline, can be incorporatedinto a peptide so that an amino acid can form a branch therewith, forexample, by forming a peptide bond to the free amino side group, fromthat residue. Alternatively, amino acids with a free carboxyl side chaingroup, such as, but not limited to, glutamic acid, aspartic acid andhomocitrulline, can be incorporated into the peptide so that an aminoacid can form a branch therewith, for example, by forming a peptide bondto the free carboxyl side group, from that residue. The amino acidforming the branch can be linked to a side chain group of an amino acidin the peptide chain by any type of covalent bond, including, but notlimited to, peptide bonds, ester bonds and disulfide bonds. In aspecific embodiment, amino acids, such as those described above, thatare capable of forming a branch point, are substituted for BLyS bindingpolypeptide residues within a peptide including a BLyS bindingpolypeptide sequence.

Branched peptides can be prepared by any method known in the art. Forexample, but not by way of limitation, branched peptides can be preparedas follows: (1) the amino acid to be branched from the main peptidechain can be purchased as an N-alpha-tert-butyloxycarbonyl (Boc)protected amino acid pentafluorophenyl (Opfp) ester and the residuewithin the main chain to which this branched amino acid will be attachedcan be an N-Fmoc-alpha-gamma-diaminobutyric acid; (2) the coupling ofthe Boc protected amino acid to diaminobutyric acid can be achieved byadding 5 grams of each precursor to a flask containing 150 ml DMF, alongwith 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing thesolution to mix for 24 hours; (3) the peptide can then be extracted fromthe 150 ml coupling reaction by mixing the reaction with 400 mldichlormethane (DCM) and 200 ml 0.12N HCl in a 1 liter separatoryfunnel, and allowing the phases to separate, saving the bottom aqueouslayer and re-extracting the top layer two more times with 200 ml 0.12NHCl; (4) the solution containing the peptide can be dehydrated by adding2-5 grams magnesium sulfate, filtering out the magnesium sulfate, andevaporating the remaining solution to a volume of about 2-5 ml; (5) thedipeptide can then be precipitated by addition of ethyl acetate and then2 volumes of hexanes and then collected by filtration and washed twotimes with cold hexanes; and (6) the resulting filtrate can belyophilized to achieve a light powder form of the desired dipeptide.Branched peptides prepared by this method will have a substitution ofdiaminobutyric acid at the amino acid position which is branched.Branched peptides containing an amino acid or amino acid analogsubstitution other than diaminobutyric acid can be prepared analogouslyto the procedure described above, using the N-Fmoc coupled form of theamino acid or amino acid analog.

In a preferred embodiment, the BLyS binding polypeptide is a cyclicpeptide. Cyclization can be, for example, but not by way of limitation,via a disulfide bond between two cysteine residues or via an amidelinkage. For example, but not by way of limitation, disulfide bridgeformation can be achieved by (1) dissolving the purified peptide at aconcentration of between 0.1-0.5 mg/ml in 0.01 M ammonium acetate, pH7.5; (2) adding to the dissolved peptide 0.01 M potassium ferricyanidedropwise until the solution appears pale yellow in color and allowingthis solution to mix for 24 hours; (3) concentrating the cyclizedpeptide to 5-10 ml of solution, repurifying the peptide by reversephase-high pressure liquid chromatography (RP-HPLC) and finallylyophilizing the peptide. In a specific embodiment, in which the peptidedoes not contain two appropriately situated cysteine residues, cysteineresidues can be introduced at the amino-terminus and/or carboxy-terminusand/or internally such that the peptide to be cyclized contains twocysteine residues spaced such that the residues can form a disulfidebridge. Alternatively, a cyclic peptide can be obtained by generating anamide linkage using, for example but not limited to, the followingprotocol: An allyl protected amino acid, such as aspartate, glutamate,asparagine or glutamine, can be incorporated into the peptide as thefirst amino acid, and then the remaining amino acids are coupled on. Theallyl protective group can be removed by a two hour mixing of thepeptide-resin with a solution of tetrakistriphenylphosphine palladium(0) in a solution of chloroform containing 5% acetic acid and 2.5%N-methylmorpholine. The peptide resin can be washed three times with0.5% N,N-diisopropylethylamine (DIEA) and 0.5% sodiumdiethyldithiocabamate in DMF. The amino terminal Fmoc group on thepeptide chain can be removed by two incubations for 15 minutes each in20% piperdine in DMF, and washed three times with DMF for 30 secondseach. The activator mix, N-methylmorpholine and HBTU in DMF, can bebrought onto the column and allowed to couple the free amino terminalend to the carboxyl group generated by removal of the allyl group tocyclize the peptide. The peptide can be cleaved from the resin asdescribed in the general description of chemical peptide synthesis aboveand the peptide purified by reverse phase-high pressure liquidchromatography (RP-HPLC). In a specific embodiment, in which the peptideto be cyclized does not contain an allyl protected amino acid, an allylprotected amino acid can be introduced into the sequence of the peptide,at the amino-terminus, carboxy-terminus or internally, such that thepeptide can be cyclized.

In addition, according to certain embodiments, it is preferable that theBLyS binding polypeptides are produced having or retaining an aminoterminal (N-terminal) and/or a carboxy terminal (C-terminal) cappinggroup, which may protect the N-terminal or C-terminal amino acid fromundesirable chemical reactions during use or which may permit furtherconjugations or manipulations of the binding polypeptide, for example,in conjugating the binding polypeptide to a chromatographic supportresin or matrix or to another peptide to tether the binding polypeptideto a resin or support. Such N-terminal and C-terminal groups may also beused to label or tag the binding polypeptide to detect bound complexesor to locate the binding polypeptide (whether bound or unbound to a BLyStarget protein) for example, at some point in a separation procedure.Accordingly, a BLyS binding polypeptide synthesized in its final formfor use in a detection or separation procedure may contain an N-terminaland/or a C-terminal capping group. A particularly preferred N-terminalcapping group, which may be present or retained in binding polypeptides,is an acetyl group (Ac). A particularly preferred C-terminal cappinggroup, which may be present or retained in binding polypeptides, is anamide group. In a further preferred embodiment, the BLyS bindingpolypeptides have an acetyl group as an N-terminal capping group and anamide group as a C-terminal capping group.

The BLyS binding polypeptides may also be prepared commercially bycompanies providing polypeptide synthesis as a service (e.g., BACHEMBioscience, Inc., King of Prussia, Pa.; Quality Controlled Biochemicals,Inc., Hopkinton, Mass.).

The nucleic acid sequence encoding a BLyS binding polypeptide can beproduced and isolated using well-known techniques in the art. In oneexample, nucleic acids encoding the BLyS binding polypeptides arechemically synthesized based on knowledge of the amino acid sequence ofthe BLyS binding polypeptide (preferably the sequence is codon optimizedto the host system in which the polypeptide will be expressed). Inanother example, nucleic acids encoding a BLyS binding polypeptide areobtained by screening an expression library (e.g., a phage displaylibrary) to identify phage expressing BLyS binding polypeptides, andisolating BLyS binding polypeptide encoding nucleic acid sequences fromthe identified library member (e.g., via polymerase chain reactionmethodology using primers flanking the polypeptide encoding sequences).

Thus, BLyS binding polypeptidess can also be obtained by recombinantexpression techniques. (See, e.g., Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d Ed., Glover, D. M. (ed.), (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989); DNA Cloning: APractical Approach (MRL Press, Ltd., Oxford, U.K., 1985), Vols. I, II.

To produce a recombinant BLyS binding polypeptide, a nucleic acidsequence encoding the BLyS binding polypeptide is operatively linked toa promoter such that the BLyS binding polypeptide is produced from saidsequence. For example, a vector can be introduced into a cell, withinwhich cell the vector or a portion thereof is expressed, producing theBLyS binding polypeptides. In a preferred embodiment, the nucleic acidis DNA if the source of RNA polymerase is DNA-directed RNA polymerase,but the nucleic acid may also be RNA if the source of polymerase isRNA-directed RNA polymerase or if reverse transcriptase is present inthe cell or provided to produce DNA from the RNA. Such a vector canremain episomal or, become chromosomally integrated, as long as it canbe transcribed to produce the desired RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be bacteriophage, plasmid, viral, retroviral, or othersknown in the art, used for replication and expression in bacterial,fungal, plant, insect or mammalian cells. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.Introduction of the vector construct into the host cell can be effectedby techniques known in the art which include, but are not limited to,calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are well known in the art andare described, for example, in many standard laboratory manuals, such asDavis et al., Basic Methods In Molecular Biology (1986).

The present invention also contemplates the use of BLyS bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, BLyS binding polypeptide fragments or variants thereof)that are recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous polypeptide(or portion thereof, preferably at least 10, at least 20, at least 30,at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90 or at least 100 amino acids of the heterologous polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. For example, BLySbinding polypeptides may be used to target heterologous polypeptides toparticular cell types (e.g., cells of monocytic lineage and B-cells),either in vitro or in vivo, by fusing or conjugating the heterologouspolypeptides to BLyS binding polypeptides that are specific forparticular cell surface antigens (e.g., membrane-bound BLyS on cells ofmonocytic lineage) or which bind antigens (i.e., BLyS) that bindparticular cell surface receptors (e.g., TACI and/or BCMA located on Bcells). BLyS binding polypeptides fused or conjugated to heterologouspolypeptides may also be used in in vitro immunoassays and purificationmethods using methods known in the art. See e.g., Harbor et al., supra,and PCT publication WO 93/2 1232; EP 439 095; Naramura et al., Immunol.Lett., 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc.Nat'l Acad. Sci. USA, 89:1428-1432 (1992); Fell et al., J. Immunol.,146:2446-2452 (1991), which are incorporated by reference in theirentireties.

The present invention further contemplates the use of compositionscomprising, or alternatively consisting of, heterologous polypeptidesfused or conjugated to BLyS binding polypeptide fragments.

Fusion proteins useful in the methods of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofBLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof), such methods can be used to generate BLyS bindingpolypeptides with altered activity (e.g., BLyS binding polypeptides withhigher affinities and lower dissociation rates). See, generally, U.S.Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, andPatten et al., Curr. Opinion Biotechnol., 8:724-33 (1997); Harayama,Trends Biotechnol., 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol.,287:265-76 (1999); and Lorenzo and Blasco, Biotechniques, 24(2):308-13(1998) (each of these patents and publications are hereby incorporatedby reference in its entirety). In one embodiment, polynucleotidesencoding BLyS binding polypeptides may be altered by being subjected torandom mutagenesis by error-prone PCR, random nucleotide insertion orother methods prior to recombination. In another embodiment, one or moreportions of a polynucleotide encoding a BLyS binding polypeptide whichportions specifically bind to BLyS may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Polypeptides of the present invention include products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses.

The BLyS binding polypeptides that are used in the methods of thepresent invention may be modified during or after synthesis ortranslation, e.g., by glycosylation, acetylation, benzylation,phosphorylation, amidation, pegylation, formylation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,ubiquitination, etc. (See, for instance, Creighton, Proteins: Structuresand Molecular Properties, 2d Ed. (W.H. Freeman and Co., N.Y., 1992);Postranslational Covalent Modification of Proteins, Johnson, ed.(Academic Press, New York, 1983), pp. 1-12; Seifter et al., Meth.Enzmmol., 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci.,663:48-62 (1992).) In specific embodiments, the peptides are acetylatedat the N-terminus and/or amidated at the C-terminus.

BLyS binding polypeptides containing two or more residues that have thepotential to interact, such as for example, two cysteine residues in apolypeptide, may be treated under oxidizing conditions or otherconditions that promote interaction of these residues (e.g, dislulfidebridge formation).

Further BLyS binding polypeptide modifications contemplated hereininclude, for example, any of numerous chemical modifications carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc. Additionalpost-translational/post-synthesis modifications that may be employedinclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression.

Chemically modified derivatives of BLyS binding polypetides may be usedwhich may provide additional advantages such as increased affinity,decreased off-rate, solubility, stability and in vivo or in vitrocirculating time of the polypeptide, or decreased immunogenicity (see,U.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any, on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol., 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides, 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem., 10:638-646 (1999), thedisclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the BLyS binding poypeptide with consideration of effects onfunctional domains of the polypeptide. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol., 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include, for example, lysineresidues and the N-terminal amino acid residues; those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. In a preferred embodiment, the polyethylene glycol moleculeis attached at an amino group, such as attachment at the N-terminus orto a lysine side chain amino group.

As suggested above, polyethylene glycol may be attached to polypeptidesvia linkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a polypeptide via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the polypeptide or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of thepolypeptide.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration, one may selectfrom a variety of polyethylene glycol molecules (by molecular weight,branching, etc.), the proportion of polyethylene glycol molecules topolypeptide molecules in the reaction mix, the type of pegylationreaction to be performed, and the method of obtaining the selectedN-terminally pegylated polypeptide. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylatedpolypeptide molecules. Selective N-terminal modification of proteins maybe accomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminus) available for derivatization in a particular protein. Underthe appropriate reaction conditions, substantially selectivederivatization of the protein at the N-terminus with a carbonyl groupcontaining polymer is achieved.

As indicated above, pegylation of the polypeptides may be accomplishedby any number of means. For example, polyethylene glycol may be attachedto the protein either directly or by an intervening linker. Linkerlesssystems for attaching polyethylene glycol to proteins are described inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys., 9:249-304 (1992);Francis et al., Intern. J. of Hematol., 68:1-18 (1998); U.S. Pat. No.4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, thedisclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of polypeptides without an intervening linker employstresylated MPEG, which is produced by the modification of monomethoxypolyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the polyeptide. Thus, the inventionincludes polypeptide-polyethylene glycol conjugates produced by reactingpolypeptides with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to polypeptides using a numberof different intervening linkers. For example, U.S. Pat. No. 5,612,460,the entire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol topolypeptides. Polypeptide-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the polypeptide by a linker can alsobe produced by reaction of polypeptides with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to polypeptides aredescribed in WO 98/32466, the entire disclosure of which is incorporatedherein by reference. Pegylated BLyS binding polypeptide productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

The number of polyethylene glycol moieties attached to each polypeptide(i.e., the degree of substitution) may also vary. For example, thepegylated polypeptides may be linked, on average, to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.Similarly, the average degree of substitution may range within rangessuch as 1-3,2-4, 3-5,4-6, 5-7,6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14,13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moietiesper polypeptide molecule. Methods for determining the degree ofsubstitution are discussed, for example, in Delgado et al., Crit. Rev.Thera. Drug Carrier Sys., 9:249-304 (1992).

BLyS Binding Polypeptide Multimers, Conjugates and Fusions

The methods of the present invention may also be carried out usingmultivalent BLyS binding polypeptides. BLyS binding polypeptides may bemonomeric, dimeric, trimeric, or higher-order multimers. In a preferredembodiment multivalent BLyS binding polypeptides are homotrimeric. Inanother preferred embodiment a homotrimeric BLyS binding polypeptidebinds a single homotrimeric BLyS.

In another preferred embodiment, monomeric or multimeric BLyS bindingpolypeptides are conjugated with another polypeptide or other chemicalcompound. For example, BLyS binding polypeptide(s) may be conjugated toa radioactive or other toxic compound so as to target and destroy cellsexpressing BLyS.

The present invention also encompasses the use of heteromeric multimerscomprised of one or more BLyS binding polypeptides and one or morenon-BLyS binding polypeptides or other chemical moieties. Suchheteromeric multimers may be monomeric, dimeric, trimeric, tetrameric,pentameric, or higher-order multimers. Heteromeric BLyS bindingmultimers may be used to target, bind, inhibit, and/or activateresponses in cells expressing BLyS and receptors for the heterologous,non-BLyS binding polypeptide or other chemical moiety. Such activatedresponses may include, for example, apoptosis or other biologically andchemically mediated forms of cell destruction. Heteromeric BLyS bindingmultimers may also be used to target BLyS expressing cells so as tointroduce a desired molecule or compound to the cells. For example, aheteromeric BLyS binding multimer may be conjugated with a radioactiveor otherwise toxic compound so as to kill BLyS expressing cells. As analternative example, a heteromeric BLyS binding and Adenovirus-bindingmultimer could be used to specifically target and introduceadenovirus-mediated gene therapeutics into BLyS expressing cells.

BLyS binding polypeptide multimers may be fused or conjugated ashomopolymers and heteropolymers using methods known in the art. In apreferred embodiment BLyS binding polypeptides are linked ashomomultimers wherein the linker or linkers provide sufficient lengthand flexibility such that each BLyS binding polypeptide maysimultaneously bind an individual BLyS molecule. In another preferredembodiment BLyS binding polypeptides are linked as heteromultimerswherein the linker or linkers provide sufficient length and flexibilitysuch that each BLyS binding polypeptide may simultaneously bindindividual BLyS molecules and the heterologous polypeptide or chemicalmoiety may simultaneously bind to its target. Numerous examples ofsuitable linker molecules are known in the art. (See, for example,Todorovska et al., J. Immunol. Methods, 248(1-2):47-66 (2001); Mehvar,J. Control Release, 69(1):1-25 (2000); Francis et. al., Int. J.Hematol., 68(1):1-18 (1998).) In specific embodiments, the linker is amember selected from the group consisting of: (a) a peptide linker; (b)a glutamate linker; and (c) a polyethylene glycol linker. The length oflinkers to be used according to the methods of the invention mayroutinely be determined using techniques known in the art. In specificembodiments, the linker is 5-60 angstroms in length. In otherembodiments, the linker is 10-50, 10-40, 10-30, or 10-20 angstroms inlength. In further embodiments, the linker is about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstromsin length. In this context “about” includes the recited length, and/orlengths that are larger or smaller by several (5, 4, 3, 2, or 1)angstroms. In other embodiments, the linker is at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100angstroms in length.

In a preferred embodiment, BLyS binding polypeptides may be fused withhuman serum albumin (HA). See, e.g., U.S. application Ser. No.09/833,245, filed Apr. 12, 2001, which is hereby incorporated byreference herein. In one embodiment, the albumin fusion proteincomprises HA as the N-terminal portion, and a BLyS binding polypeptideas the C-terminal portion. In another embodiment the albumin fusionprotein comprise HA as the C-terminal portion, and a BLyS bindingpolypeptide as the N-terminal portion.

In other embodiments, the albumin fusion protein has a BLyS bindingpolypeptide fused to both the N-terminus and the C-terminus of albumin.In one preferred embodiment, the BLyS binding polypeptides fused at theN- and C-termini are the same BLyS binding polypeptides. In anotherpreferred embodiment, the BLyS binding polypeptides fused at the N- andC-termini are different BLyS binding polypeptides. In another preferredembodiment, a BLyS binding polypeptide is fused at either the N- orC-terminus of HA and a different (non-BLyS binding) polypeptide is fusedat either the C- or N-terminus, respectively.

In addition to albumin fusion proteins in which the BLyS bindingpolypeptide(s) is (are) fused to the N-terminus and/or C-terminus of HA,BLyS binding polypeptide/albumin fusion proteins may also be produced byinserting the BLyS binding polypeptide into an internal region orregions of HA. For instance, within the protein sequence of the HAmolecule a number of loops or turns exist between the end and beginningof α-helices, which are stabilized by disulphide bonds (see FIGS. 9-11in U.S. application Ser. No. 09/833,245). The loops, as determined fromthe crystal structure of HA (FIG. 13 of U.S. application Ser. No.09/833,245) (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and1UOR) for the most part extend away from the body of the molecule. Theseloops are useful for the insertion, or internal fusion, oftherapeutically active peptides (particularly those requiring asecondary structure to be functional) or therapeutic proteins, toessentially generate an albumin molecule with specific biologicalactivity.

Loops in human albumin structure into which binding polypeptides may beinserted to generate albumin fusion proteins include: Val54-Asn61,Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His 247-Glu252, Glu266-Glu277, Glu 280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475,Thr478-Pro486, and Lys560-Thr566. In more preferred embodiments,polypeptides are inserted into the Val54-Asn61, Gln170-Ala176, and/orLys560-Thr566 loops of mature human serum albumin (SEQ ID NO:445).

In specific embodiments, BLyS binding polypeptides are attached tomacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, topolypeptides. In a preferred embodiment, the radiometal ion associatedwith the macrocyclic chelators attached to BLyS binding polypeptides is¹¹¹In. In another preferred embodiment, the radiometal ion associatedwith the macrocyclic chelator attached to BLyS binding polypeptides is⁹⁰Y. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the BLyS bindingpolypeptides via a linker molecule. Examples of linker molecules usefulfor conjugating DOTA to a polypeptide are commonly known in the art—see,for example, DeNardo et al., Clin. Cancer Res., 4(10):2483-90 (1998);Peterson et al., Bioconjug. Chem., 10(4):553-7 (1999); and Zimmerman etal, Nucl. Med. Biol., 26(8):943-50 (1999), which are hereby incorporatedby reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361and 5,756,065, which disclose chelating agents that may be conjugated toantibodies, and methods for making and using them, are herebyincorporated by reference in their entireties. Though U.S. Pat. Nos.5,652,361 and 5,756,065 focus on conjugating chelating agents toantibodies, one skilled in the art would be readily able to adapt themethod disclosed therein in order to conjugate chelating agents to otherpolypeptides.

The BLyS binding polypeptides can be recovered and purified by knownmethods which include, but are not limited to, ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

The BLyS binding polypeptides may also be modified with a detectablelabel, including, but not limited to, an enzyme, prosthetic group,fluorescent material, luminescent material, bioluminescent material,radioactive material, positron emitting metal, nonradioactiveparamagnetic metal ion, and affinity label for detection and isolationof BLyS target. The detectable substance may be coupled or conjugatedeither directly to the polypeptides or indirectly, through anintermediate (such as, for example, a linker known in the art) usingtechniques known in the art. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase,glucose oxidase or acetylcholinesterase; examples of suitable prostheticgroup complexes include streptavidin/biotin and avidin/biotin; examplesof suitable fluorescent materials include biotin, umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include a radioactivemetal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or otherradioisotopes such as, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In,¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon(¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ^(0.47)Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷C,⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

In specific embodiments, BLyS binding polypetides are attachedmacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. Inspecific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the BLyS bindingpolypeptide via a linker molecule. Examples of linker molecules usefulfor conjugating DOTA to a polypeptide are commonly known in the art—see,for example, DeNardo et al., Clin. Cancer Res., 4(10):2483-90 (1998);Peterson et al., Bioconjug. Chem., 10(4):553-7 (1999); and Zimmerman etal, Nucl. Med. Biol., 26(8):943-50 (1999), which are hereby incorporatedby reference in their entirety.

In a specific embodiment, BLyS binding polypeptides are labeled withbiotin.

The present invention further encompasses the use of BLyS bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, BLyS binding polypeptide fragments or variants thereof),conjugated to a diagnostic or therapeutic agent. The BLyS bindingpolypeptides can be used diagnostically to, for example, monitor orprognose the development or progression of a tumor as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the BLyS bindingpolypeptide to a detectable substance. Examples of detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions suchas, for example, those described herein. The detectable substance may becoupled or conjugated either directly to the BLyS binding polypeptide orindirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to BLySbinding polypeptides for use as diagnostics according to the presentinvention.

Further, a BLyS binding polypeptide (including a molecule comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof), may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters such as, for example,²¹³Bi, or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I,⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include, but are not limited to, paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease,RNAse, and puromycin and frragments, variants or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Techniques known in the art may be applied to label BLyS bindingpolypeptides. Such techniques include, but are not limited to, the useof bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

The BLyS binding polypeptides which are conjugates can be used formodifying a given biological response, the therapeutic agent or drugmoiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, but are not limited to, for example, a toxin such as abrin,ricin A, alpha toxin, pseudomonas exotoxin, or diphtheria toxin,saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin andcholera toxin; a protein such as tumor necrosis factor,alpha-interferon, beta-interferon, nerve growth factor, platelet derivedgrowth factor, tissue plasminogen activator, an apoptotic agent, e.g.,TNF-alpha, TNF-beta, AIM I (see, International Publication No. WO97/33899), AIM II (see, International Publication No. WO 97/34911), fasligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (see,International Publication No. WO 99/23105), a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin or endostatin; or, biologicalresponse modifiers such as, for example, lymphokines, interleukin-1(IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), or other growth factors.

A BLyS binding polypeptide (including a molecule comprising, oralternatively consisting of, a BLyS binding polypeptide fragment orvariant thereof), with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Characterization of BLyS Binding Polypeptides

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) may be characterized in a variety of ways. Inparticular, BLyS binding polypeptides and related molecules may beassayed for the ability to specifically bind to BLyS or a fragment ofBLyS (e.g., to the soluble form or the membrane-bound form of BLyS)using techniques described herein or routinely modifying techniquesknown in the art. BLyS or BLyS fragments that may be specifically boundby the compositions useful according to the invention include, but arenot limited to, human BLyS (SEQ ID NOs:173 and/or 174) or BLyS expressedon human monocytes; murine BLyS (SEQ ID NOs:175 and/or 176) or BLySexpressed on murine monocytes; rat BLyS (either the soluble forms asgiven in SEQ ID NOs:177, 178, 179 and/or 180 or in a membrane associatedform, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., themonkey BLyS polypeptides of SEQ ID NOS:181 and/or 182, the soluble formof monkey BLyS, or BLyS expressed on monkey monocytes) or fragmentsthereof. Preferably compositions useful according to the invention bindhuman BLyS (SEQ ID NOs:173 and/or 174) or fragments thereof. Assays forthe ability of the BLyS binding polypeptides to specifically bind BLySor a fragment of BLyS may be performed in solution (e.g., Houghten,Bio/Techniques, 13:412-421(1992)), on beads (e.g., Lam, Nature,354:82-84 (1991)), on chips (e.g., Fodor, Nature, 364:555-556 (1993)),on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat.Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull etal., Proc. Natl. Acad. Sci. USA, 89:1865-1869 (1992)) or on phage (e.g.,Scott and Smith, Science, 249:386-390 (1990); Devlin, Science,249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA,87:6378-6382 (1990); and Felici, J. Mol. Biol., 222:301-310 (1991))(each of these references is incorporated herein in its entirety byreference). BLyS binding polypeptides that have been identified tospecifically bind to BLyS or a fragment of BLyS can then be assayed fortheir specificity and affinity for BLyS or a fragment of BLyS using orroutinely modifying techniques described herein or otherwise known inthe art.

The BLyS binding polypeptides may be assayed for specific binding toBLyS and cross-reactivity with other BLyS-like polypeptides by anymethod known in the art. In particular, the ability of a BLyS bindingpolypeptide to specifically bind to the soluble form or membrane-boundform of BLyS and the specificity of the BLyS binding polypeptide,fragment, or variant for BLyS polypeptide from a particular species(e.g., murine, monkey or human, preferably human) may be determinedusing or routinely modifying techniques described herein or otherwiseknown in art.

Assays which can be used to analyze specific binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”assays, “immunoprecipitation” assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, radiometric assays, and fluorescentassays, to name but a few. Such assays are routine and well known in theart (see, e.g., Current Protocols in Molecular Biology, Vol. 1, Ausubelet al, eds. (John Wiley & Sons, Inc., New York 1994), which isincorporated by reference herein in its entirety) and could easily beadapted to make use of a BLyS binding polypeptide (possibly inconjunction with an anti-BLyS binding polypeptide antibody) in place ofan anti-BLyS antibody. Exemplary immunoassays that could be modified touse a BLyS binding polypeptide are described briefly below (but are notintended by way of limitation).

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), incubating the membranewith BLyS binding polypeptide (the BLyS binding polypeptide of interest)diluted in blocking buffer, washing the membrane in washing buffer,incubating the membrane with a secondary antibody (which recognizes theBLyS binding polypeptide) conjugated to an enzyme (e.g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., ³²Por ¹²⁵I) diluted in blocking buffer, washing the membrane in washbuffer, and detecting the presence of the antigen. Alternatively, theBLyS binding polypeptide may be directly conjugated to a detectionmolecule (e.g., an enzyme or radiolabel), thereby omitting the need fora secondary anti-BLyS binding polypeptide antibody. One of skill in theart would be knowledgeable as to the parameters that can be modified toincrease the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., CurrentProtocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John Wiley& Sons, Inc., New York 1994) at 10.8.1.

ELISAs comprise preparing antigen (e.g., BLyS target), coating the wellof a 96-well microtiter plate with the antigen, washing away antigenthat did not bind the wells, adding the BLyS binding polypeptide ofinterest conjugated to a detectable compound such as an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) to the wells andincubating for a period of time, washing away unbound BLyS bindingpolypeptides or non-specifically bound BLyS binding polypeptides, anddetecting the presence of the BLyS binding polypeptides specificallybound to the antigen coating the well. In ELISAs the BLyS bindingpolypeptide employed in the assay does not have to be conjugated to adetectable compound; instead, an antibody that recognizes the BLySbinding polypeptide and that is conjugated to a detectable compound maybe added to the well. Further, instead of coating the well with theantigen, the BLyS binding polypeptide may be coated to the well. In thiscase, the detectable molecule could be the antigen conjugated to adetectable compound such as an enzyme (e.g., horseradish peroxidase oralkaline phosphatase). One of skill in the art would be knowledgeable asto the parameters that can be modified to increase the signal detectedas well as other variations of ELISAs known in the art. For furtherdiscussion regarding ELISAs see, e.g., Current Protocols in MolecularBiology, Vol. 1, Ausubel et al, eds. (John Wiley & Sons, Inc., New York1994) at 11.2.1.

Immunoprecipitation protocols generally use antibody molecules toimunopreciptate a protein of interest. A BLyS preciptation protocolcould easily be modified to use a BLyS binding polypeptide in place ofan anti-BLyS antibody. Immunopreciptation protocols generally compriselysing a population of cells in a lysis buffer such as RIPA buffer (1%NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented withprotein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,aprotinin, sodium vanadate), adding the antibody of interest to the celllysate, incubating for a period of time (e.g., 1 to 4 hours) at 40degrees C., adding protein A and/or protein G sepharose beads to thecell lysate, incubating for about an hour or more at 40 degrees C.,washing the beads in lysis buffer and resuspending the beads inSDS/sample buffer. If one wanted to substitute a BLyS bindingpolypeptide for the anti-BLyS antibody one could readily do so, and thenisolate the BLyS-BLyS binding polypeptide complexes with an antibodythat recognizes the BLyS binding polypeptide. Then the triple complex ofBLyS, BLyS binding polypeptide, and anti-BLyS binding polypeptideantibody could be isolated using protein A and/or Protein G as describedabove. Such a protocol may be desirable if, for example, the anti-BLySbinding polypeptide antibody has a higher affinity for the BLyS bindingpolypeptide than the anti-BLyS antibody may have for BLyS.

The effectiveness of incorporating a BLyS binding polypeptide in animmunoprecipitation protocol to precipitate BLyS can be assessed by,e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the BLyS binding polypeptide to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Current Protocols in Molecular Biology, Vol. 1, Ausubel et al,eds. (John Wiley & Sons, Inc., New York 1994) at 10.16.1.

The binding affinity of a BLyS binding polypeptide (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) to an antigen and the off-rate of an BLySbinding polypeptide-antigen interaction can be determined by competitivebinding assays. One example of a competitive binding assay is a modifiedradioimmunoassay comprising the incubation of labeled antigen (e.g., ³H-or ¹²⁵I-labeled BLyS target) with the BLyS binding polypeptide ofinterest in the presence of increasing amounts of unlabeled antigen,followed by detection of the BLyS binding polypeptide bound to thelabeled antigen. The affinity of the BLyS binding polypeptide of thepresent invention for BLyS and the binding off-rates can be determinedfrom the data by Scatchard plot analysis. Competition with an anti-BLySantibody or BLyS binding polypeptide can also be determined usingradioimmunoassays. In this case, BLyS is incubated with a BLyS bindingpolypeptide of the present invention conjugated to a labeled compound(e.g., with ³H or ¹²⁵I) in the presence of increasing amounts of anunlabeled BLyS binding polypeptide or anti-BLyS antibody.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof) to BLyS, or fragments ofBLyS. BIAcore kinetic analysis comprises analyzing the binding anddissociation of BLyS from chips with immobilized BLyS bindingpolypeptides on their surface (see Example 6, infra).

The BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) can also be assayed for their ability to inhibit,increase, or not significantly alter, the binding of BLyS to a BLySreceptor (e.g., TACI and BCMA) using techniques known to those skilledin the art. For example, cells expressing a receptor for BLyS (e.g.,IM9, REH, ARH-77 cells, Namalwa, and RPMI-8226 B cell tumor lines aswell as peripheral CD20+ B cells) can be contacted with BLyS in thepresence or absence of a BLyS binding polypeptide, and the ability ofthe BLyS binding polypeptide to inhibit, increase, or not significantlyalter, BLyS binding to the cells can be measured. Alternatively, theBLyS binding polypeptide may be preincubated with the BLyS prior toexposure of the BLyS to cells expressing the BLyS receptor. BLyS bindingto cells can be measured by, for example, flow cytometry or ascintillation assay. BLyS or the BLyS binding polypeptide can be labeledwith a detectable compound such as a radioactive label (e.g., ³²P, ³⁵S,and ¹²⁵I) or a fluorescent label (e.g., fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine) to enable detection of an interaction between BLySand a BLyS receptor and/or BLyS and a BLyS binding polypeptide.

The ability of BLyS binding polypeptides to inhibit, increase, or notsignificantly alter, BLyS binding to a BLyS receptor can also bedetermined in cell-free assays. For example, native or recombinant BLyS(e.g., having the amino acid sequence of amino acids 134-285 of SEQ IDNO:173) or a fragment thereof can be contacted with a BLyS bindingpolypeptide and the ability of the BLyS binding polypeptide to inhibit,increase, or not significantly alter, BLyS from binding to a BLySreceptor can be determined. Preferably, the BLyS binding polypeptide orBLyS receptor is immobilized on a solid support and BLyS or a BLySfragment is labeled with a detectable compound. Alternatively, BLyS or aBLyS fragment is immobilized on a solid support and the BLyS bindingpolypeptide is labeled with a detectable compound. BLyS may be partiallyor completely purified (e.g., partially or completely free of otherpolypeptides) or part of a cell lysate. Further, the BLyS polypeptidemay be a fusion protein comprising BLyS or a biologically active portionthereof and a domain such as an Immunoglobulin Fc orglutathionine-S-transferase. Additionally, the BLyS binding polypeptideand/or BLyS receptor may be a fusion protein comprising a BLyS bindingportion of the polypeptide or receptor and a domain such as anImmunoglobulin Fc or glutathionine-S-transferase. For example, aminoacid residues 1-154 of TACI (GenBank accesion number AAC51790), or 1-48of BCMA (GenBank accession number NP_(—)001183) may be fused to the Fcregion of an IgG molecule and used in a cell free assay to determine theability of BLyS binding polypeptides to inhibit, increase, or notsignificantly alter, BLyS binding to a BLyS receptor. Alternatively,BLyS can be biotinylated using techniques well known to those skilled inthe art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).

The BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof), can also be assayed for their ability to inhibit,stimulate, or not significantly alter, BLyS-induced B-cell proliferationusing techniques known to those of skill in the art. For example, B-cellproliferation can be assayed by ³H-thymidine incorporation assays andtrypan blue cell counts (see, e.g., Moore et al., Science, 285: 260-263(1999)). Further, the BLyS binding polypeptides, or fragments orvariants thereof, can be assayed for their ability to inhibit,stimulate, or not significantly alter, BLyS-induced activation ofcellular signaling molecules and transcription factors such ascalcium-modulator and cyclophilin ligand (CAML), calcineurin, nuclearfactor of activated T cells transcription factor (NF-AT), nuclearfactor-kappa B (NF-kappa B), SRF, activator protein-1 (AP-1),extracellular-signal regulated kinase 1 (ERK-1), polo like kinases(PLK), ELF-1, high mobility group I (HMG-I), and/or high mobility groupY (HMG-Y) using techniques known to those of skill in the art (see,e.g., von Bulow and Bram, Science, 278:138-141(1997)). For example,NF-AT activity can be determined by electromobility gel shift assays, bydetecting the expression of a protein known to be regulated by NF-AT(e.g., IL-2 expression), by detecting the induction of a reporter gene(e.g., an NF-AT regulatory element operably linked to a nucleic acidencoding a detectable marker such as luciferase, beta-galactosidase orchloramphenicol acetyltransferase (CAT)), or by detecting a cellularresponse (e.g., cellular differentiation, or cell proliferation).

The BLyS binding polypeptides, or fragments or variants thereof can alsobe assayed for their ability to neutralize, enhance, or notsignificantly alter, BLyS activity. For example, BLyS bindingpolypeptides or fragments or variants thereof, may be routinely testedfor their ability to inhibit BLyS from binding to cells expressing thereceptor for BLyS.

Uses of the Binding Polypeptides and Recombinant Bacteriophage

The BLyS binding polypeptides described herein are especially useful todetect, isolate, or remove BLyS target proteins in solutions. Suchsolutions may be simple dispersions or solutions of BLyS and/orBLyS-like polypeptide in water or aqueous buffer or more complexsolutions, such as, a blood and other biological fluids, tissuehomogenates cell extracts, or biopsy samples, and cell culture mediacontaining BLyS or BLyS-like polypeptides. Biological fluids include,but are not limited to sera, plasma, lymph, blood, blood fractionsurine, synovial fluid, spinal fluid, saliva, and mucous.

In one embodiment, the present invention provides a method for detectinga BLyS protein and/or a BLyS-like polypeptide in a solution comprisingcontacting the solution with a BLyS binding polypeptide and detectingbinding of BLyS or BLyS-like polypeptide to the BLyS bindingpolypeptide. The BLyS binding polypeptide may be either free orimmobilized. Preferably, the BLyS binding polypeptide is a polypeptideimmobilized on a solid surface or chromatographic material or the wellof a plastic microtiter assay dish.

Another embodiment of the present invention is a method for isolatingBLyS protein and/or BLyS-like polypeptide from a solution containing it,comprising:

-   -   (a) contacting the solution with a BLyS binding polypeptide        under conditions that permit binding of BLyS and/or BLyS-like        polypeptides to BLyS binding polypeptide, and    -   (b) recovering the BLyS and/or BLyS-like polypeptides.

A further embodiment of the present invention is a method for isolatingBLyS protein and/or BLyS-like polypeptide from a solution containing it,comprising:

-   -   (a) contacting the solution with a BLyS binding polypeptide        under conditions that permit binding of BLyS and/or BLyS-like        polypeptides to BLyS binding polypeptide, and    -   (b) separating the complex(es) formed by the BLyS binding        polypeptide and BLyS and/or BLyS-like polypeptides from other        components of the solution.

Preferably such method also includes the further steps of:

-   -   (c) dissociating the BLyS binding polypeptide from the BLyS        and/or BLyS-like polypeptides, and    -   (d) recovering the dissociated, BLyS and/or BLyS-like        polypeptide.

The invention also provides for the use of kits containing a bindingpolypeptide for use in methods of detecting or isolating BLyS and/orBLyS-like polypeptides.

According to the invention, detection or isolation of BLyS targetproteins comprises contacting a solution containing a BLyS targetprotein with a BLyS binding polypeptide. Depending on the particularapplication, the BLyS binding polypeptide may be free in solution orimmobilized on a solid support or chromatographic material. Sufficienttime is allowed to permit binding between the BLyS target protein andthe binding polypeptides, and non-binding components in the solution ormixture are removed or washed away. The formation of a binding complexbetween the binding polypeptide and the BLyS target protein can then bedetected, for example, by detecting the signal from a label on thebinding polypeptide, which is one component of the binding complex. Alabel may be any label that generates a signal that can be detected bystandard methods, such as a fluorescent label, a radioactive compound,or an enzyme that reacts with a substrate to generate a detectablesignal. Suitable such labels are discussed above. A phage bindingpolypeptide according to the invention, that is, a recombinant phagedisplaying a BLyS binding polypeptide on its surface, may form a complexwith BLyS and/or BLyS-like polypeptides that is detectable as aprecipitate or sediment in a reaction tube, which can be detectedvisually after settling or centrifugation. Alternatively, asandwich-type assay may be used, wherein a BLyS binding polypeptide isimmobilized on a solid support such as a plastic tube or well, or achromatographic support matrix such as agarose beads, then the solutionsuspected of containing the BLyS target is contacted with theimmobilized binding polypeptide and non-binding materials or componentsare removed or washed away.

The binding polypeptides according to this invention are particularlyuseful for detection and/or isolation of BLyS and/or BLyS-likepolypeptides by affinity chromatography methods. Any conventional methodof chromatography may be employed. Preferably, a BLyS bindingpolypeptide will be immobilized on a solid support suitable, forexample, for packing a chromatography column. The immobilized BLySbinding polypeptide affinity ligand can then be loaded or contacted witha feed stream under conditions favorable to formation of bindingpolypeptide/BLyS (or BLyS-like polypeptide) complexes. Non-bindingmaterials can be washed away. Examples of suitable wash conditions canreadily be determined by one of skill in the art and include but are notlimited to [PBS/0.01% Tween 20, pH7.2] and [1M NaCl/10 mM Tris, pH7.5].Tris wash buffers may be preferable since phosphates can preciptate in50% ethylene glycol. In general, non-limiting terms, wash buffers arepH7.0, optionally containing 0.0 to 1.5 M NaCl, more preferably 1 MNaCl. Additionally, wash buffers may optionally contain a milddetrgenet, such as, for example, Tween 20, Tween 80, or NP-80. BLyS orBLyS-like polypeptide can be eluted from the BLyS binding polypeptide byintroducing solution conditions that favor dissociation of the bindingcomplex. Suitable elution solutions can readily be determined by one ofskill in the art and include but are not limited to [50% ethylmeglycol/100 mM NaOAc]. By way of non-limiting example, useful elutionbuffers, for the purposes of the present invention contain 40-60%ethylene glycol, preferably 50% ethylene glycol.; and 50-100 mM NaOAcwith a pH in the range of pH 4-pH7, more preferably, pH 4-pH 6 and mostpreferably pH 4.5-pH 5.5. Preferably, a fast flow affinitychromatographic technique is used to bind the molecules and from whichpurified BLyS or BLyS-like polypeptides are eluted.

Alternatively, batch chromatography can be carried out by mixing asolution containing the BLyS target and the BLyS binding polypeptide,then isolating complexes of the BLyS target and the bindingpolypeptides. For this type of separation, many methods are known. Forexample, the binding polypeptide may be immobilized on a solid supportsuch as beads, then separated from the feed stream along with the BLyStarget by filtration. In another example, the BLyS binding polypeptidemay be modified with its own affinity tag, such as a polyHis tail orstreptavidin binding region, which can be used to isolate the bindingpolypeptide after complexes have formed using an immobilized metalaffinity chromatographic resin or steptavidin-coated substrate. Onceseparated, the BLyS target can be released from the binding polypeptideunder elution conditions and recovered in a purified form.

Methods of producing BLyS or a BLyS-like polypeptides usually yield BLySor BLyS-like polypeptides in a feed stream that additionally containsimpurities (with respect to the BLyS target). One purpose of the presentinvention is to produce BLyS binding polypeptides and preparations (suchas affinity chromatography media or surfaces) comprising BLyS bindingpolypeptides that allow rapid and highly specific purification of BLyStarget proteins from a feed stream. BLyS binding polypeptides obtainedherein may easily be tailored to isolate BLyS target protein from aparticular feed stream, using or routinely modifying conditions andtechniques known in the art. If an alternate production method for BLySis used, producing a different feed stream, a different set of BLySbinding polypeptides and/or conditions may be necessary to achieve thesame level of purification. The new set of BLyS binding polypeptidesand/or conditions can be readily obtained following or modifyingprocedures outlined herein, or otherwise known in the art.

Use of BLyS Binding Polypeptides for Epitope Mapping

The present invention provides BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof), that can be used to identifyepitopes of BLyS. In particular, the BLyS binding polypeptides of thepresent invention can be used to identify epitopes of human BLyS (SEQ IDNOs:173 and/or 174) or BLyS expressed on human monocytes; murine BLyS(SEQ ID NOs:175 and/or 176) or BLyS expressed on murine monocytes; ratBLyS (either the soluble forms as given in SEQ ID NOs:177, 178, 179and/or 180 or in a membrane associated form, e.g., on the surface of ratmonocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ IDNOS:181 and/or 182, the soluble form of monkey BLyS, or BLyS expressedon monkey monocytes) using techniques described herein or otherwiseknown in the art. Fragments which function as epitopes may be producedby any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.USA, 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211.)

Diagnostic Uses of BLyS Binding Polypeptides

Labeled and non-labelled BLyS binding polypeptides (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) which specifically bind to BLyS can beused for diagnostic purposes to detect, diagnose, prognose, or monitordiseases and/or disorders associated with the aberrant expression and/oractivity of BLyS or BLyS receptor. The invention provides for thedetection of aberrant expression of BLyS comprising: (a) assaying theexpression of BLyS in a biological sample from an individual using oneor more BLyS binding polypeptides that specifically binds to BLyS; and(b) comparing the level of BLyS with a standard level of BLyS, e.g., innormal biological samples, whereby an increase or decrease in theassayed level of BLyS compared to the standard level of BLyS isindicative of aberrant expression.

By “biological sample” is intended any fluids and/or cells obtained froman individual, body fluid, body tissue, body cell, cell line, tissueculture, or other source which may contain BLyS protein or mRNA. Bodyfluids include, but are not limited to, sera, plasma, urine, synovialfluid, spinal fluid, saliva, and mucous. Tissues samples may be takenfrom virtually any tissue in the body. Tissue samples may also beobtained from autopsy material. Methods for obtaining tissue biopsiesand body fluids from mammals are well known in the art. Where thebiological sample is to include mRNA, a tissue biopsy is the preferredsource.

The invention also provides for the detection of aberrant expression ofBLyS receptor comprising (a) assaying the expression of BLyS receptor ina biological sample from an individual using one or more BLyS bindingpolypeptides or fragments or variants thereof that specifically bindsonly to soluble BLyS, but does not inhibit BLyS/BLyS receptor binding.Such a BLyS binding polypeptide, by way of an example that is not to beconstrued as limiting, would be one that is able to capture abiotinylated BLyS from solution, but that would not prevent BLyS frombinding to it receptor expressed, for example on IM-9 cells, and (b)comparing the level of BLyS receptor with a standard level of BLySreceptor, e.g., in normal tissue or cell samples, whereby an increase ordecrease in the assayed level of BLyS receptor compared to the standardlevel of BLyS receptor is indicative of aberrant expression.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) which specifically bind to BLyS can be used fordiagnostic purposes to detect, diagnose, prognose, or monitor immunesystem diseases and disorders, including but not limited to autoimmunediseases and disorders and/or immunodeficiencies, and/or diseases,disorders, or conditions associated therewith. The invention providesfor the detection of aberrant expression of BLyS comprising: (a)assaying the expression of BLyS in a biological sample from anindividual using one or more BLyS binding polypeptides that specificallybinds to BLyS; and (b) comparing the level of BLyS with a standard levelof BLyS, e.g., in normal biological samples, whereby an increase ordecrease in the assayed level of BLyS compared to the standard level ofBLyS is indicative of an autoimmune disorder or disease and/or animmunodeficiency. In specific embodiments, an increase in the assayedlevel of BLyS is indicative of an autoimmune disorder or disease. Inother specific embodiments, a decrease in the assayed level of BLyS isindicative of an immunodeficiency.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) which specifically bind to BLyS but do not inhibitBLyS/BLys receptor binding can be used for diagnostic purposes todetect, diagnose, prognose, or monitor immune system diseases anddisorders, including but not limited to autoimmune diseases anddisorders and/or immunodeficiencies, and/or diseases, disorders, orconditions associated therewith. The invention provides for thedetection of aberrant expression of BLyS receptor comprising: (a)assaying the expression of BLyS receptor in a biological sample from anindividual using one or more BLyS binding polypeptides that specificallybinds to BLyS; and (b) comparing the level of BLyS receptor with astandard level of BLyS receptor, e.g., in normal biological samples,whereby an increase or decrease in the assayed level of BLyS receptorcompared to the standard level of BLyS receptor is indicative of anautoimmune disorder or disease and/or an immunodeficiency. In specificembodiments, an increase in the assayed level of BLyS receptor isindicative of an autoimmune disorder or disease. In other specificembodiments, a decrease in the assayed level of BLyS receptor isindicative of an immunodeficiency.

Autoimmune disorders, diseases, or conditions that may be detected,diagnosed, prognosed, or monitored using the BLyS binding polypeptidesinclude, but are not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia,antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy),multiple sclerosis, neuritis, uveitis ophthalmia, polyendocrinopathies,purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-ManSyndrome, autoimmune pulmonary inflammation, myocarditis, IgAglomerulonephritis, dense deposit disease, rheumatic heart disease,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis), systemic lupus erhythematosus, discoidlupus, Goodpasture's syndrome, Pemphigus, receptor autoimmunities suchas, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)insulin resistance, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, rheumatoid arthritis, schleroderma withanti-collagen BLyS binding polypeptides, mixed connective tissuedisease, polymyositis/dermatomyositis, pernicious anemia, idiopathicAddison's disease, infertility, glomerular nephritis such as primaryglomerular nephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders.

In specific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing, and/or monitoringdiseases or disorders associated with hypergammaglobulinemia (e.g.,AIDS, autoimmune diseases, and some immunodeficiencies). In otherspecific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing, and/or monitoringdiseases or disorders associated with hypogammaglobulinemia (e.g., animmunodeficiency).

Immunodeficiencies that may be detected, diagnosed, prognosed, ormonitored using the BLyS binding polypeptides include, but are notlimited to, severe combined immunodeficiency (SCID)-X linked,SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),X-linked agammaglobulinemia (XLA), Bruton's disease, congenitalagammaglobulinemia, X-linked infantile agammaglobulinemia, acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

Elevated levels of soluble BLyS have been observed in the serum ofpatients with Systemic Lupus Erythematosus (SLE). In comparing the seraof 150 SLE patients with that of 38 control individuals, it was foundthat most of the SLE patients had more than 5 ng/ml of serum BLyS, morethan 30% of SLE patients had levels greater than 10 ng/ml, andapproximately 10% of SLE patients had serum BLyS levels greater than 20ng/ml. In contrast, the majority of normal controls had BLyS levels lessthan 5 ng/ml, and less than 10% had levels higher than 10 ng/ml. Theelevated levels of BLyS protein in sera is present in the soluble formand has biologic activity as assayed by the ability to stimulateanti-IgM treated B cells in vitro. SLE patients with more than 15 ng/mlserum BLyS were also found to have elevated levels of anti-dsDNAantibodies compared to both normal controls and SLE patients with lessthan 5 ng/ml of serum BLyS (unpublished data).

In addition the serum of two subgroups of patients which were positivefor anti-nuclear antibodies (ANA+) but did not meet the formalrequirements of the American College of Rheumatology (ACR) forclassification of SLE were anaylzed for BLyS levels. The first subgroupof sera was ANA+ sera that came from patients who did not present withthe clinical impression of SLE. This group had only slightly elevatedlevels of BLyS (˜9 ng/ml BLyS). The second subgroup, however, which wasANA+ sera from patients who presented with the clinical impression ofSLE, had significantly increased BLyS levels (˜15 ng/ml). These resultssuggest that an elevated level of BLyS precedes the formal fulfillmentof the ACR criteria. The ACR criteria are desrcibed in Tan et al.,Arthritis and Rheumatism, 25:1271-1277 (1982).

Thus, in specific embodiments, BLyS binding polypeptides whichspecifically bind to BLyS can be used for diagnostic purposes to detect,diagnose, prognose, or monitor Systemic Lupus Erythematosus orconditions associated therewith. The invention provides for thedetection of aberrant expression of BLyS comprising: (a) assaying theexpression of BLyS in a biological sample (e.g., serum, synovial fluid)of an individual using one or more BLyS binding polypeptides thatspecifically binds to BLyS; and (b) comparing the level of BLyS with astandard level of BLyS, e.g., in normal biological samples, whereby anincrease in the assayed level of BLyS compared to the standard level ofBLyS is indicative of SLE.

In additional embodiments, BLyS binding polypeptides which specificallybind to BLyS can be used for diagnostic purposes to detect, diagnose,prognose, or monitor Rheumatoid Arthritis. The invention provides forthe detection of aberrant expression of BLyS comprising: (a) assayingthe expression of BLyS in a biological sample (e.g., serum, synovialfluid) of an individual using one or more BLyS binding polypeptides thatspecifically binds to BLyS; and (b) comparing the level of BLyS with astandard level of BLyS, e.g., in normal biological samples, whereby anincrease in the assayed level of BLyS compared to the standard level ofBLyS is indicative of Rheumatoid Arthritis.

In specific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing and/or prognosing diseases ordisorders of cells of hematopoietic origin. Cells of hematopoieticorigin include, but are not limited to, lymphocytes (e.g., B cells and Tcells), monocytes, macrophages, dendritic cells, polymorphonuclearleukocytes (e.g., basophils, eosinophils, neutrophils), mast cells,platelets, erythrocytes and progenitor cells of these lineages. Cells ofhematopoietic origin include, but are not limited to, healthy anddiseased cell as found present in an animal, preferably a mammal andmost preferably a human, or as isolated from an animal, transformedcells, cell lines derived from the above listed cell types, and cellcultures derived from the above listed cell types. Cells ofhematopoietic origin may be found or isolated in, for example, resting,activated or anergic states.

In specific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing and or monitoringgrowth, progression, and/or metastases of malignancies and proliferativediseases or disorders associated with increased cell survival, or theinhibition of apoptosis. For a review of such disorders, see Fishman etal., Medicine, 2d Ed. (J. B. Lippincott Co., Philadelphia 1985). Anextensive list of examples of proliferative diseases and disorders ispresented below in the section of this application entitled “TherapeuticUses of BLyS Binding Polypeptides.” Proliferative diseases and disordersis also extended to include premalignant conditions (e.g., benigntumors, hyperproliferative disorders, and benign proliferativedisorders—see below) as well as proliferative disorders of B cells,monocytes, macrophages, and T cells. Other abnormal growth conditionsthat may be treated, diagnosed, prognosed or monitored include, but arenot limited to, hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, Basic Pathology, 2d Ed. (W.B. Saunders Co., Philadelphia1976), pp. 68-79.) Hyperplasia is a form of controlled cellproliferation involving an increase in cell number in a tissue or organ,without significant alteration in structure or function. As but oneexample, endometrial hyperplasia often precedes endometrial cancer.Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplasia can occur in epithelial or connective tissue cells.Atypical metaplasia involves a somewhat disorderly metaplasticepithelium. Dysplasia is frequently a forerunner of cancer, and is foundmainly in the epithelia; it is the most disorderly form ofnon-neoplastic cell growth, involving a loss in individual celluniformity and in the architectural orientation of cells. Dysplasticcells often have abnormally large, deeply stained nuclei, and exhibitpleomorphism. Dysplasia characteristically occurs where there existschronic irritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

In preferred embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing and or monitoringgrowth, progression, and/or metastases of malignancies and proliferativediseases or disorders of monocytic cells.

In specific embodiments, the present invention encompasses methods andcompositions for detecting, diagnosing, prognosing and or monitoringgrowth, progression, and/or metastases of malignancies and proliferativediseases or disorders of B cells.

The invention provides a diagnostic assay for diagnosing or prognosing adisease or disorder, comprising: (a) assaying for the level of BLyS in abiological sample of an individual using one or more BLyS bindingpolypeptides that specifically bind to BLyS; and (b) comparing the levelof BLyS with a standard BLyS level, e.g., in a biological sample from apatient without the disease or disorder, whereby an increase or decreasein the assayed BLyS level compared to the standard level of BLyS isindicative of a particular disease or disorder. With respect to cancer,the presence of a relatively high amount of BLyS in biopsied tissue froman individual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

In specific embodiments, the presence of a relatively high amount ofmembrane-bound BLyS in a biological sample is indicative of monocyticcell related leukemias or lymphomas, such as, for example acutemyelogenous leukemia, and/or the severity thereof.

In other specific embodiments, the presence of a relatively high amountof BLyS receptor in a biological sample (as determined using BLySbinding polypeptides that bind to soluble BLyS, but do not inhibitBLyS/BLyS receptor binding) is indicative of B cell related leukemias orlymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma,non-Hodgkin's lymphoma, and Hodgkin's disease), and/or the severitythereof.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) can be used to assay protein levels in a biologicalsample using classical immunohistological methods as described herein oras known to those of skill in the art (e.g., see Jalkanen et al., J.Cell. Biol., 101:976-985 (1985); Jalkanen et al., J. Cell. Biol.,105:3087-3096 (1987)). Other methods that can be used for detectingprotein gene expression that might utilize BLyS binding polypeptides orfragments or variants thereof include, but are not limited to, theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase, alkaline phophatase, andhorseradish peroxidase; radioisotopes, such as iodine (¹²¹I, ¹²³I, ¹²⁵I,¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In,¹¹³In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon(¹³³Xe), fluorine (¹⁸F), ^(15f3)Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

Certain embodiments of the invention are directed to the detection anddiagnosis of a disease or disorder associated with aberrant expressionof BLyS or BLyS receptor in an animal, preferably a mammal and mostpreferably a human. In one embodiment, diagnosis comprises: (a)administering (for example, parenterally, subcutaneously, orintraperitoneally) to a subject an effective amount of a labeled BLySbinding polypeptide (including molecules comprising, or alternativelyconsisting of, BLyS binding polypeptide fragments or variants thereof)that specifically binds to BLyS; (b) waiting for a time intervalfollowing the administering for permitting the labeled BLyS bindingpolypeptide to preferentially concentrate at sites in the subject whereBLyS is expressed (and for unbound labeled molecule to be cleared tobackground level); (c) determining background level; and (d) detectingthe labeled BLyS binding polypeptide in the subject, such that detectionof labeled BLyS binding polypeptide or fragment thereof above thebackground level and above or below the level observed in a personwithout the disease or disorder indicates that the subject has aparticular disease or disorder associated with aberrant expression ofBLyS or BLyS receptor. Background level can be determined by variousmethods, including comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

It will be understood by those skilled in the art that the size of thesubject and the imaging system used will determine the quantity ofimaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ⁹⁹Tc. Thelabeled BLyS binding polypeptide will then preferentially accumulate atthe location of cells which contain the specific protein. In vivo tumorimaging is described in Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments,” Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment for monitoring of the disease or disorder, the methodis carried out by repeating the method for diagnosing the disease ordisorder, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc. andcomparing the results of the successive tests.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (see, e.g., Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Immunophenotyping Using BLyS Binding Polypeptides

The BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) may be utilized for immunophenotyping of cell linesand biological samples by their BLyS expression or BLyS receptorexpression. Various techniques can be employed utilizing BLyS bindingpolypeptides, fragments, or variants to screen for cellular populations(i.e., immune cells, particularly monocytic cells or B-cells) expressingBLyS or BLyS receptor. Such techniques include magnetic separation usingBLyS binding polypeptide-coated magnetic beads, “panning” with BLySbinding polypeptide attached to a solid matrix (i.e., plate), and flowcytometry (see, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.,minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

In one embodiment, BLyS binding polypeptides (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) are used to identify cells of monocyticor B cell origin.

Therapeutic Uses of BLyS Binding Polypeptides

The present invention is further directed to BLyS bindingpolypeptide-based therapies which involve administering BLyS bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, BLyS binding polypeptide fragments or variants thereof)to an animal, preferably a mammal, and most preferably a human, patientfor treating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, BLyS binding polypeptides and nucleic acids encoding BLySbinding polypeptides and antibodies that bind BLyS binding polypeptidesas described herein. The BLyS binding polypeptides can be used to treat,ameliorate or prevent diseases, disorders or conditions associated withaberrant expression and/or activity of BLyS or BLyS receptor, including,but not limited to, any one or more of the diseases, disorders, orconditions described herein. The treatment and/or prevention ofdiseases, disorders, or conditions associated with aberrant BLySexpression and/or activity or aberrant BLyS receptor expression and/oractivity includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. BLyS bindingpolypeptides may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein.

BLyS binding polypeptides of the present invention (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) that function as agonists or antagonistsof BLyS, preferably of BLyS-induced signal transduction, can beadministered to an animal to treat, prevent or ameliorate a disease ordisorder associated with aberrant BLyS expression, lack of BLySfunction, aberrant BLyS receptor expression, or lack of BLyS receptorfunction. For example, BLyS binding polypeptides which disrupt theinteraction between BLyS and one or more of its receptors may beadministered to an animal to treat, prevent or ameliorate a disease ordisorder associated with aberrant BLyS expression, excessive BLySfunction, aberrant BLyS receptor expression, or excessive BLyS receptorfunction. BLyS binding polypeptides which do not prevent BLyS frombinding its receptor but inhibit or downregulate BLyS-induced signaltransduction can be administered to an animal to treat, prevent orameliorate a disease or disorder associated with aberrant BLySexpression, excessive BLyS function, aberrant BLyS receptor expression,or excessive BLyS receptor function. In particular, BLyS bindingpolypeptides of the present invention which prevent BLyS-induced signaltransduction by specifically recognizing the unbound BLyS,receptor-bound BLyS, or both unbound and receptor-bound BLyS can beadministered to an animal to treat, prevent or ameliorate a disease ordisorder associated with aberrant BLyS expression, excessive BLySfunction, aberrant BLyS receptor expression, or excessive BLyS receptorfunction.

The ability of a BLyS binding polypeptide to inhibit or downregulateBLyS-induced signal transduction may be determined by techniquesdescribed herein or otherwise known in the art. For example,BLyS-induced receptor activation and the activation of signalingmolecules can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or a signaling molecule byimmunoprecipitation followed by western blot analysis (for example, asdescribed herein).

In a specific embodiment, a BLyS binding polypeptide of the presentinvention (including molecules comprising, or alternatively consistingof, BLyS binding polypeptide fragments or variants thereof) thatinhibits or reduces BLyS activity by at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to BLySactivity in the absence of the BLyS binding polypeptide, is administeredto an animal to treat, prevent or ameliorate a disease or disorderassociated with aberrant BLyS expression, excessive BLyS function,aberrant BLyS receptor expression, or excessive BLyS receptor function.In another embodiment, a combination of BLyS binding polypeptides, acombination of BLyS binding polypeptide fragments, a combination of BLySbinding polypeptide variants, or a combination of BLyS bindingpolypeptides, BLyS binding polypeptide fragments, and/or variants thatinhibit or reduce BLyS activity by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 65%, at least60%, at least 55%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to BLyS activity in absence of said BLyS bindingpolypeptides, BLyS binding polypeptide fragments, and/or BLyS bindingpolypeptide variants are administered to an animal to treat, prevent orameliorate a disease or disorder associated with aberrant BLySexpression, excessive BLyS function, aberrant BLyS receptor expression,or excessive BLyS receptor function.

Further, BLyS binding polypeptides of the present invention (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof) which activate BLyS-inducedsignal transduction can be administered to an animal to treat, preventor ameliorate a disease or disorder associated with aberrant BLySexpression, lack of BLyS function, aberrant BLyS receptor expression, orlack of BLyS receptor function. These BLyS binding polypeptides maypotentiate or activate either all or a subset of the biologicalactivities of BLyS-mediated receptor activation, for example, byinducing multimerization of BLyS and/or multimerization of the receptor.The BLyS binding polypeptides may be administered with or without beingpre-complexed with BLyS. In a specific embodiment, a BLyS bindingpolypeptide of the present invention that increases BLyS activity by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or100% or more relative to BLyS activity in absence of the BLyS bindingpolypeptide is administered to an animal to treat, prevent or amelioratea disease or disorder associated with aberrant BLyS expression, lack ofBLyS function, aberrant BLyS receptor expression, or lack of BLySreceptor function. In another embodiment, a combination of BLyS bindingpolypeptides, a combination of BLyS binding polypeptide fragments, acombination of BLyS binding polypeptide variants, or a combination ofBLyS binding polypeptides, BLyS binding polypeptide fragments and/orBLyS binding polypeptide variants that increase BLyS activity by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or100% or more relative to BLyS activity in absence of the said BLySbinding polypeptides or BLyS binding polypeptide fragments and/or BLySbinding polypeptide variants is administered to an animal to treat,prevent or ameliorate a disease or disorder associated with aberrantBLyS expression, lack of BLyS function, aberrant BLyS receptorexpression, or lack of BLyS receptor function.

In a specific embodiment, the present invention provides a method oftreating, preventing or ameliorating a disease or disorder associatedwith aberrant BLyS or BLyS receptor expression or activity, comprisingadministering to an animal in which such treatment, prevention oramelioration is desired, a BLyS binding polypeptide in an amounteffective to treat, prevent or ameliorate the disease or disorder.Diseases and disorders which may be treated, prevented or ameliorated bythis method include, but are not limited to, immune system diseases anddisorders (e.g., autoimmune diseases and disorders, immunodeficiencies,lupus, rheumatoid arthritis, multiple sclerosis, hypogammaglobulinemiaand hypergammaglobulinemia), graft vs. host disease, proliferativediseases and disorders (e.g., cancer) and infectious diseases anddisorders.

In a specific embodiment, the present invention provides a method oftreating, preventing or ameliorating a disease or disorder of cells ofhematopoietic origin, comprising administering to an animal in whichsuch treatment, prevention, or amelioration is desired, a BLyS bindingpolypeptide in an amount effective to treat, prevent or ameliorate thedisease or disorder. Cells of hematopoietic origin include, but are notlimited to, lymphocytes (e.g., B cells and T cells), monocytes,macrophages, dendritic cells, polymorphonuclear leukocytes (e.g.,basophils, eosinophils, neutrophils), mast cells, platelets,erythrocytes and progenitor cells of these lineages.

One or more BLyS binding polypeptides of the present invention(including molecules comprising, or alternatively consisting of, BLySbinding polypeptide fragments or variants thereof) that specificallybind to BLyS may be used locally or systemically in the body as atherapeutic. The BLyS binding polypeptides (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) may also be advantageously utilized incombination with monoclonal or chimeric antibodies, lymphokines and/orhematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), forexample, which serve to increase the number or activity of effectorcells which interact with the BLyS binding polypeptides.

The BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) may be administered alone or in combination with othertypes of treatments (e.g., radiation therapy, chemotherapy, hormonaltherapy, immunotherapy, anti-tumor agents, anti-angiogenesis andanti-inflammatory agents).

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing BLyS binding polypeptides (including moleculescomprising, or alternatively consisting of, BLyS binding polypeptidefragments or variants thereof) that specifically bind to BLyS, orpolynucleotides encoding BLyS binding polypeptides that specificallybind to BLyS, for both immunoassays directed to and therapy of disordersrelated to BLyS polynucleotides or polypeptides, including fragmentsthereof. Such BLyS binding polypeptides will preferably have an affinityfor BLyS and/or BLyS fragments. Preferred binding affinities includethose with a dissociation constant or K_(D) of less than or equal to5×10 ⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵M. More preferably, BLyS binding polypeptides bind BLyS target proteinswith a dissociation constant or K_(D) less than or equal to 5×10⁻⁶ M,10⁻⁶ M, 5×10 ⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even more preferably,BLyS binding polypeptides bind BLyS target proteins with a dissociationconstant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10 ⁻¹² M, 10⁻¹² M, 5×10 ⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In a preferred embodiment, BLyS binding polypeptides neutralize BLySactivity. In another preferred embodiment, BLyS binding polypeptidesinhibit B cell proliferation.

In a preferred embodiment, BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof) inhibit or reduce binding ofthe soluble form of BLyS to a BLyS receptor. In another preferredembodiment BLyS binding polypeptides inhibit or reduce B cellproliferation induced by the soluble form of BLyS. In another preferredembodiment BLyS binding polypeptides inhibit or reduce immunoglobulinproduction induced by the soluble form of BLyS.

In a preferred embodiment, BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof) inhibit or reduce binding ofmembrane-bound BLyS to a BLyS receptor. In another preferred embodiment,BLyS binding polypeptides inhibit or reduce B cell proliferation inducedby the membrane-bound form of BLyS. In another preferred embodiment,BLyS binding polypeptides inhibit or reduce immunoglobulin productioninduced by the membrane bound form of BLyS.

In a preferred embodiment, BLyS binding polypeptides (includingmolecules comprising, or alternatively consisting of, BLyS bindingpolypeptide fragments or variants thereof) inhibit or reduce binding ofboth the soluble and membrane-bound forms of BLyS to a BLyS receptor. Inanother preferred embodiment, BLyS binding polypeptides inhibit orreduce B cell proliferation induced by either or both forms of BLyS. Inanother preferred embodiment, BLyS binding polypeptides inhibit orreduce immunoglobulin production induced by either or both forms ofBLyS.

In one embodiment, the invention provides a method of deliveringradiolabelled BLyS binding polypeptide and/or BLyS binding polypeptideconjugates to targeted cells, such as, for example, monocytic cellsexpressing the membrane-bound form of BLyS, or B cells expressing a BLySreceptor.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing immunoglobulin production (e.g., IgM, IgG, and/orIgA production), comprising, or alternatively consisting of, contactingan effective amount of BLyS binding polypeptide with BLyS, wherein theeffective amount of BLyS binding polypeptide inhibits or reduces BLySmediated immunoglobulin production. In another embodiment, the inventionprovides methods and compositions for inhibiting or reducingimmunoglobulin production (e.g., IgM, IgG, and/or IgA production),comprising, or alternatively consisting of, administering to an animalin which such inhibition or reduction is desired, a BLyS bindingpolypeptide in an amount effective to inhibit or reduce immunoglobulinproduction.

In another embodiment, the invention provides methods and compositionsfor stimulating immunoglobulin production (e.g., IgM, IgG, and/or IgAproduction), comprising, or alternatively consisting of, contacting aneffective amount of BLyS binding polypeptide with BLyS, wherein theeffective amount of the BLyS binding polypeptide stimulates BLySmediated immunoglobulin production. In another embodiment, the inventionprovides methods and compositions for stimulating immunoglobulinproduction (e.g., IgM, IgG, and/or IgA production) comprising, oralternatively consisting of, administering to an animal in which suchstimulation is desired, a BLyS binding polypeptide in an amounteffective to stimulate immunoglobulin production. Determination ofimmunoglobulin levels are most often performed by comparing the level ofimmunoglobulin in a sample to a standard containing a known amount ofimmunoglobulin using ELISA assays. Determination of immunoglobulinlevels in a given sample, can readily be determined using ELISA or othermethod known in the art.

Receptors belonging to the TNF receptor (TNFR) super family (e.g., TACIand BCMA) can be classified into two types based on the presence orabsence of a conserved cytoplasmic domain responsible for apoptosiscalled a “death domain.” TNF receptors without death domains, such asTNF-R2 HVEM/ATAR, RANK, CD27, CD30, CD40, and OX40 interact with TNFreceptor associated factors (TRAF 1-6) and mediate anti-apoptoticsurvival and or proliferative responses via activation of thetranscription factor NF-kappaB (reviewed in Wajant et al., Cytokine andGrowth Factor Reviews, 10(1):15-26, 1999). TACI and BCMA do not containdeath domains.

Investigation of BLyS induced signaling in human tonsillar B cellsco-stimulated with Staph. aureus Cowan consistently revealed that mRNAfor ERK-1 and PLK were upregulated by BLyS+SAC treatment (see Example12). Polo like kinases (PLK) belong to a sub family of serine/threoninekinases related to Saccharomyces cerevisiae cell cycle protein CDC5(29). The expression of PLK is induced during G2 and S phase of the cellcycle. PLK is reported to play a role in cell proliferation (Lee et al.,Proc. Natl. Acad. Sci., 95:9301-9306, 1998). The role orextracellular-signal related kinases (ERK1/2) in cell survival andproliferative effects of growth factors and other agonists has beenextensively studied. The induced expression of PLK and ERK-1 isconsistent with the survival and proliferative effects of BLyS on Bcells.

Additionally, in some samples of human tonsillar B cells stimulated withBLys and SAC, mRNA for CD25 (IL-2Ralpha) was upregulated. Nuclearextracts from Human tonsillar B cells treated with BLyS and from IM-9cells treated with BLyS were able to shift probes from the CD25 promoterregion containing sites for NF-kappaB, SRF, ELF-1 and HMGI/Y in anelectromobility shift assay. ELF-1 for example, is a transcriptionfactor that is part of the ETS family of proteins and whose expressionappears to be restricted to T and B cells. Binding sites for ELF-1 havebeen described in the promoters of a number of proteins that areimportant in the regulation of the immune response.

Thus BLyS induced signaling has been shown to be consistent with theactivation of cellular activation and cellular proliferation pathways aswell as with cellular signaling pathways that regulate B cell lifespan.Further, BLyS treatment of B cells induces cellular proliferationimmunoglobulin secretion, a characteristic of activated B cells (Mooreet al., Science, 285:260-263, 1999). BLyS binding polypeptides complexedwith BLyS may inhibit, stimulate, or not significantly alter these BLySmediated activities.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing B cell proliferation, comprising, oralternatively consisting of, contacting an effective amount of BLySbinding polypeptide with BLyS, wherein the effective amount of BLySbinding polypeptide inhibits or reduces BLyS mediated B cellproliferation. In another embodiment, the invention provides methods andcompositions for inhibiting or reducing B cell proliferation comprising,or alternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a BLyS binding polypeptide in anamount effective to inhibit or reduce B cell proliferation.

In one embodiment, the invention provides methods and compositions forstimulating B cell proliferation, comprising, or alternativelyconsisting of, contacting an effective amount of BLyS bindingpolypeptide with BLyS, wherein the effective amount of BLyS bindingpolypeptide stimulates BLyS mediated B cell proliferation.

In one embodiment, the invention provides methods and compositions forstimulating B cell proliferation, comprising, or alternativelyconsisting of, administering to an animal in which such stimulation isdesired, a BLyS binding polypeptide in an amount effective to stimulateB cell proliferation.

B cell proliferation is most commonly assayed in the art by measuringtritiated thymidine incorporation (see Examples 7 and 8). This and otherassays are commonly known in the art and may be routinely adapted forthe use of determining the effect of BLys binding polypeptides on B cellproliferation.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing activation of B cells, comprising, oralternatively consisting of, contacting an effective amount of BLySbinding polypeptide with BLyS, wherein the effective amount of BLySbinding polypeptide inhibits or reduces BLyS mediated B cell activation.

In one embodiment, the invention provides methods and compositions forinhibiting or reducing activation of B cells, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, a BLyS binding polypeptide in anamount effective to inhibit or reduce B cell activation.

In one embodiment, the invention provides methods and compositions forincreasing activation of B cells, comprising, or alternativelyconsisting of, contacting an effective amount of BLyS bindingpolypeptide with BLyS, wherein the effective amount of BLyS bindingpolypeptide increases BLyS mediated activation of B cells.

In one embodiment, the invention provides methods and compositions forincreasing activation of B cells, comprising, or alternativelyconsisting of, administering to an animal in which such increase isdesired, a BLyS binding polypeptide in an amount effective to increase Bcell activation.

B cell activation can measured in a variety of ways, such as FACSanalysis of activation markers expressed on B cells. B cells activationmarkers include, but are not limited to, CD26, CD 28, CD 30, CD 38, CD39, CD 69, CD70 CD71, CD 77, CD 83, CD126, CDw130, and B220.Additionally, B cell activation may be measured by analysis of theactivation of signaling molecules involved in B cell activation. By wayof non-limiting example, such analysis may take the form of analyzingmRNA levels of signaling molecules by Northern analysis or real time PCR(Example 12). One can also measure, for example, the phosphorylation ofsignaling molecules using anti-phosphotyrosine antibodies in a Westernblot. B cell activation may also be measured by measuring the calciumlevels in B cells. These and other methods of determining B cellactivation are commonly known in the art and may be routinely adaptedfor the use of determining the effect of BLys binding polypeptides on Bcell activation.

In one embodiment, the invention provides methods and compositions fordecreasing lifespan of B cells, comprising, or alternatively consistingof, contacting an effective amount of BLyS binding polypeptide withBLyS, wherein the effective amount of BLyS binding polypeptide inhibitsor reduces BLyS regulated lifespan of B cells.

In one embodiment, the invention provides methods and compositions fordecreasing lifespan of B cells, comprising, or alternatively consistingof, administering to an animal in which such decrease is desired, a BLySbinding polypeptide in an amount effective to decrease B cell lifespan.

In one embodiment, the invention provides methods and compositions forincreasing lifespan of B cells, comprising, or alternatively consistingof, contacting an effective amount of BLyS binding polypeptide withBLyS, wherein the effective amount of BLyS binding polypeptide increasesBLyS regulated lifespan of B cells.

In one embodiment, the invention provides methods and compositions forincreasing lifespan of B cells, comprising, or alternatively consistingof, administering to an animal in which such increase is desired, a BLySbinding polypeptide in an amount effective to increase lifespan of Bcells.

B cell life span in vivo may be measured by 5-bromo-2′-deoxyuridine(BrdU) labeling experiments which are well known to one skilled in theart. BrdU is a thymidine analogue that gets incorporated into the DNA ofdividing cells. Cells containing BrdU in their DNA can be detectedusing, for example fluorescently labeled anti-BrdU antibody and flowcytometry. Briefly, an animal is injected with BrdU in an amountsufficient to label developing B cells. Then, a sample of B cells iswithdrawn from the animal, for example, from peripheral blood, andanalyzed for the percentage of cells that contain BrdU. Such an analysisperformed at several time points can be used to calculate the half lifeof B cells. Alternatively, B cell survival may be measured in vitro. Forexample B cells may be cultured under conditions where proliferationdoes not occur, (for example the media should contain no reagents thatcrosslink the immunoglobulin receptor, such as anti-IgM antibodies) fora period of time (usually 2-4 days). At the end of this time, thepercent of surviving cells is determined, using for instance, the vitaldye Trypan Blue, or by staining cells with propidium iodide or any otheragent designed to specifically stain apoptotic cells and analyzing thepercentage of cells stained using flow cytometry. One could perform thisexperiment under several conditions, such as B cells treated with BLyS,B cells treated with BLyS/BLys binding polypeptide complexes, anduntreated B cells in order to determine the effects of BLyS and BLySbinding polypeptides on B cells survival. These and other methods fordetermining B cell lifespan are commonly known in the art and couldroutinely be adapted to determining the effect of BLyS bindingpolypeptides on BLyS regulated B cell lifespan.

In one embodiment, the invention provides a method for the specificdelivery of BLyS binding polypeptides and BLyS binding polypeptideconjugates to cells by administering molecules that are associated withheterologous polypeptides or nucleic acids. In one example, theinvention provides a method for delivering a therapeutic protein intothe targeted cell. In another example, the invention provides a methodfor delivering a single stranded nucleic acid (e.g., antisense orribozymes) or double stranded nucleic acid (e.g., DNA that can integrateinto the cell's genome or replicate episomally and that can betranscribed) in the targeted cell.

In another embodiment, the invention provides for a method of killingcells of hematopoietic origin, comprising, or alternatively consistingof, contacting BLyS binding polypeptides with BLyS to form a complex;and contacting the complex with cells of hematopoietic origin. Inspecific embodiments, the method of killing cells of hematopoieticorigin, comprises, or alternatively consists of, administering to ananimal in which such killing is desired, a BLyS binding polypeptide inan amount effective to kill cells of hematopoietic origin. Cells ofhematopoietic origin include, but are not limited to, lymphocytes (e.g.,B cells and T cells), monocytes, macrophages, dendritic cells,polymorphonuclear leukocytes (e.g., basophils, eosinophils,neutrophils), mast cells, platelets, erythrocytes and progenitor cellsof these lineages. Cells of hematopoietic origin include, but are notlimited to, healthy and diseased cell as found present in an animal,preferably a mammal and most preferably a human, or as isolated from ananimal, transformed cells, cell lines derived from the above listed celltypes, and cell cultures derived from the above listed cell types. Cellsof hematopoietic origin may be found or isolated in, for example,resting, activated or anergic states.

In another embodiment, the invention provides a method for the specificdestruction (i.e., killing) of cells (e.g., the destruction of tumorcells) by administering BLyS binding polypeptides or BLyS bindingpolypeptide conjugates (e.g., radiolabeled BLyS binding polypetidesand/or BLyS binding polypeptides conjugated with radioisotopes, toxins,or cytotoxic prodrugs). In a specific embodiment, the invention providesa method for the specific destruction of cells of monocytic lineage(e.g., monocytic cell related leukemias or lymphomas, such as, forexample acute myelogenous leukemia) by administering BLyS bindingpolypeptides or BLyS binding polypeptide conjugates (e.g., BLyS bindingpolypeptides conjugated with radioisotopes, toxins, or cytotoxicprodrugs) that specifically bind the membrane-bound form of BLyS. Inanother specific embodiment, the invention provides a method for thespecific destruction of cells of B cell lineage (e.g., B cell relatedleukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiplemyeloma, non-Hodgkin's lymphoma, and Hodgkin's disease) by administeringBLyS binding polypeptides or BLyS binding polypeptide conjugates (e.g.,BLyS binding polypeptides conjugated with radioisotopes, toxins, orcytotoxic prodrugs) that bind soluble BLyS, but do not inhibit BLySbinding to a BLyS receptor on B cells.

In another embodiment of the invention, therapeutic or pharmaceuticalcompositions are administered to an animal to treat, prevent orameliorate diseases and disorders of the immune system. In a specificembodiment, the invention provides a method of treating, preventing, orameliorating an immune system disease or disorder, comprising, oralternatively consisting of, administering to an animal in which suchtreatment, prevention, or amelioration is desired, a BLyS bindingpolypeptide in an amount effective to treat, prevent, or ameliorate theimmune system disease or disorder. Diseases and disorders of the immunesystem include, but are not limited to, autoimmune diseases anddisorders (e.g., arthritis, graft rejection, Hashimoto's thyroiditis,insulin-dependent diabetes, lupus, rheumatoid arthritisidiopathicthrombocytopenic purpura, systemic lupus erythramatosus and multiplesclerosis, and other autoimmune diseases or disorders named ordesdcribed herein), hypogammaglobulinemia, hypergammaglobulinemia,elective IgA deficiency, ataxia-telangiectasia, immunodeficiencies(e.g., common variable immunodeficiency (CVID), X-linkedagammaglobulinemia, severe combined immunodeficiency (SCID), andWiskott-Aldrich syndrome), graft vs. host disease, idiopathichyper-eosinophilic syndrome, monocytic leukemoid reaction, monocyticleukocytosis, monocytic leukopenia, monocytopenia, monocytosis, graft ortransplant rejection, as well as infectious diseases (e.g., AIDS andhepatitis).

As discussed herein, BLyS binding polypeptides and BLyS bindingpolypeptide compositions, may be used to treat, prevent, ameliorate,diagnose or prognose various immune system-related disorders and/orconditions associated with these disorders, in mammals, preferablyhumans. Many autoimmune disorders result from inappropriate recognitionof self as foreign material by immune cells. This inappropriaterecognition results in an immune response leading to the destruction ofthe host tissue. Therefore, the administration of BLyS bindingpolypeptides and BLyS binding polypeptide compositions that can inhibitan immune response, particularly the proliferation of B cells and/or theproduction of immunoglobulins, may be an effective therapy in treatingand/or preventing autoimmune disorders. Thus, in preferred embodiments,BLyS binding polypeptides and BLyS binding polypeptide compositions areused to treat, prevent, ameliorate, diagnose and/or prognose anautoimmune disorder, or condition(s) associated with such disorder.

Autoimmune disorders and conditions associated with these disorders thatmay be treated, prevented, ameliorated, diagnosed and/or prognosedaccording to the invention with the therapeutic and pharmaceuticalcompositions described herein include, but are not limited to,autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmune neutropenia,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis,neuritis, uveitis ophthalmia, polyendocrinopathies, purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis,dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome,insulin dependent diabetes mellitis, and autoimmune inflammatory eyedisease.

Additional autoimmune disorders and conditions associated with thesedisorders that may be treated, prevented, ameliorated, diagnosed and/orprognosed according to the present invention with the therapeutic andpharmaceutical compositions described herein include, but are notlimited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto'sthyroiditis) (often characterized, e.g., by cell-mediated and humoralthyroid cytotoxicity), systemic lupus erhythematosus (oftencharacterized, e.g., by circulating and locally generated immunecomplexes), discoid lupus, Goodpasture's syndrome (often characterized,e.g., by anti-basement membrane antibodies), Pemphigus (oftencharacterized, e.g., by epidermal acantholytic antibodies), Receptorautoimmunities such as, for example, (a) Graves' Disease (oftencharacterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis(often characterized, e.g., by acetylcholine receptor antibodies), and(c) insulin resistance (often characterized, e.g., by insulin receptorantibodies), autoimmune hemolytic anemia (often characterized, e.g., byphagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenicpurpura (often characterized, e.g., by phagocytosis ofantibody-sensitized platelets.H

Additional autoimmune disorders and conditions associated with thesedisorders that may be treated, prevented, ameliorated, diagnosed and/orprognosed according to the present invention with the therapeutic andpharmaceutical compositions described herein include, but are notlimited to, rheumatoid arthritis (often characterized, e.g., by immunecomplexes in joints), schleroderma with anti-collagen antibodies (oftencharacterized, e.g., by nucleolar and other nuclear antibodies), mixedconnective tissue disease (often characterized, e.g., by antibodies toextractable nuclear antigens (e.g., ribonucleoprotein)),polymyositis/dermatomyositis (often characterized, e.g., by nonhistoneANA), pernicious anemia (often characterized, e.g., by antiparietalcell, microsomes, and intrinsic factor antibodies), idiopathic Addison'sdisease (often characterized, e.g., by humoral and cell-mediated adrenalcytotoxicity, infertility (often characterized, e.g., byantispermatozoal antibodies), glomerulonephritis (often characterized,e.g., by glomerular basement membrane antibodies or immune complexes)such as primary glomerulonephritis and IgA nephropathy, bullouspemphigoid (often characterized, e.g., by IgG and complement in basementmembrane), Sjogren's syndrome (often characterized, e.g., by multipletissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetesmillitus (often characterized, e.g., by cell-mediated and humoral isletcell antibodies), and adrenergic drug resistance (including adrenergicdrug resistance with asthma or cystic fibrosis) (often characterized,e.g., by beta-adrenergic receptor antibodies), chronic active hepatitis(often characterized, e.g., by smooth muscle antibodies), primarybiliary cirrhosis (often characterized, e.g., by mitchondrialantibodies), other endocrine gland failure (often characterized, e.g.,by specific tissue antibodies in some cases), vitiligo (oftencharacterized, e.g., by melanocyte antibodies), vasculitis (oftencharacterized, e.g., by Ig and complement in vessel walls and/or lowserum complement), post-MI (often characterized, e.g., by myocardialantibodies), cardiotomy syndrome (often characterized, e.g., bymyocardial antibodies), urticaria (often characterized, e.g., by IgG andIgM antibodies to IgE), atopic dermatitis (often characterized, e.g., byIgG and IgM antibodies to IgE), asthma (often characterized, e.g., byIgG and IgM antibodies to IgE), inflammatory myopathies, and many otherinflammatory, granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, therapeutic and pharmaceutical compositionsare used to treat, prevent, ameliorate, diagnose or prognose, a memberof the group: autoimmune hemolytic anemia, as primaryglomerulonephritis, IgA glomerulonephritis, Goodpasture's syndrome,idiopathic thrombocytopenia, Multiple Sclerosis, Myasthenia Gravis,Pemphigus, polymyositis/dermatomyositis, relapsing polychondritis,rheumatoid arthritis, Sjogren's syndrome, systemic lupus erhythematosus,Uveitis, vasculitis, and primary biliary cirrhosis.

In another specific preferred embodiment, therapeutic and pharmaceuticalcompositions are used to treat, prevent, amelioate, diagnose orprognose, rheumatoid arthritis and/or medical conditions associatedtherewith.

In a specific preferred embodiment, therapeutic and pharmaceuticalcompositions are used to treat, prevent, amelioate, diagnose orprognose, lupus and/or medical conditions associated therewith.Lupus-associated conditions that may be treated, prevented, ameliorated,prognosed and/or diagnosed with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia,and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies,and anti-Sm antibodies), rashes, photosensitivity, oral ulcers,arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus(pleurisy)), renal disorders (e.g., nephritis), neurological disorders(e.g., seizures, peripheral neuropathy, CNS related disorders),gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In apreferred embodiment, therapeutic and pharmaceutical compositions areused to treat, prevent, ameliorate, diagnose, or prognose, renaldisorders associated with systemic lupus erythematosus. In a mostpreferred embodiment, therapeutic and pharmaceutical compositions areused to treat, prevent, ameliorate, diagnose, or prognose, nephritisassociated with systemic lupus erythematosus. In another most preferredembodiment, therapeutic or pharmaceutical compositions are administeredto an animal to treat, prevent or ameliorate lupus or glomerularnephritis.

In another embodiment, therapeutic or pharmaceutical compositions areadministered to an animal to treat, prevent or ameliorate anIgE-mediated allergic reaction or histamine-mediated allergic reaction.Examples of allergic reactions include, but are not limited to, asthma,rhinitis, eczema, chronic urticaria, and atopic dermatitis. In anotherembodiment, therapeutic or pharmaceutical compositions are administeredto an animal to treat, prevent, or ameliorate anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility. In another embodiment, therapeutic or pharmaceuticalcompositions are administered to an animal to treat, prevent orameliorate or modulate inflammation or an inflammatory disorder.Examples of chronic and acute inflammatory disorders that may be treatedprevented or ameliorated with the therapeutic and pharmaceuticalcompositions include, but are not limited to, chronic prostatitis,granulomatous prostatitis and malacoplakia, inflammation associated withinfection (e.g., septic shock, sepsis, or systemic inflammatory responsesyndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality,arthritis, complement-mediated hyperacute rejection, nephritis, cytokineor chemokine induced lung injury, Crohn's disease, inflammatory boweldisease, chronic and acute inflammatory pulmonary diseases, bacterialinfection, psoriasis, septicemia, cerebral malaria, arthritis,gastroenteritis, and glomerular nephritis.

In another embodiment, therapeutic or pharmaceutical compositions areadministered to an animal to treat, prevent or ameliorate ischemia andarteriosclerosis. Examples of such disorders include, but are notlimited to, reperfusion damage (e.g., in the heart and/or brain) andcardiac hypertrophy.

Therapeutic or pharmaceutical compositions may also be administered tomodulate blood clotting and to treat or prevent blood clottingdisorders, such as, for example, antibody-mediated thrombosis (i.e.,antiphospholipid antibody syndrome (APS)). For example, therapeutic orpharmaceutical compositions as described herein may be used to inhibitthe proliferation and differentiation of cells involved in producinganticardiolipin antibodies. These compositions can be used to treat,prevent, ameliorate, diagnose, and/or prognose thrombotic related eventsincluding, but not limited to, stroke (and recurrent stroke), heartattack, deep vein thrombosis, pulmonary embolism, myocardial infarction,coronary artery disease (e.g., antibody-mediated coronary arterydisease), thrombosis, graft reocclusion following cardiovascular surgery(e.g., coronary arterial bypass grafts, recurrent fetal loss, andrecurrent cardiovascular thromboembolic events.

Therapeutic or pharmaceutical compositions containing BLyS bindingpolypeptides may also be administered to treat, prevent, or ameliorateorgan rejection or graft-versus-host disease (GVHD) and/or conditionsassociated therewith. Organ rejection occurs by host immune celldestruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.Administration of BLyS binding polypeptides that inhibit an immuneresponse may be an effective therapy in preventing organ rejection orGVHD. In specific embodiments the present invention provides a method ofinhibiting or reducing graft rejection, comprising administering to ananimal in which such inhibition or reduction is desired, a BLyS bindingpolypeptide in an amount effective to inhibit or reduce graft rejection.

In another embodiment, therapeutic or pharmaceutical compositions areadministered to an animal to treat, prevent or ameliorate a disease ordisorder diseases associated with increased apoptosis including, but notlimited to, AIDS, neurodegenerative disorders (such as Alzheimer'sdisease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration), myelodysplastic syndromes (such asaplastic anemia), ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), toxin-induced liver disease(such as that caused by alcohol), septic shock, cachexia and anorexia.In another embodiment, therapeutic or pharmaceutical compositions areadministered to an animal to treat, prevent or ameliorate bone marrowfailure, for example, aplastic anemia and myelodysplastic syndrome.

In other embodiment, therapeutic or pharmaceutical compositions asdescribed herein are used to treat or prevent a proliferative disorder(e.g., cancer). In preferred embodiments, therapeutic or pharmaceuticalcompositions as described herein are used to treat or preventproliferative disorders of monocytic cells. In other preferredembodiments, therapeutic or pharmaceutical compositions as describedherein are used to treat or prevent a proliferative disorders of B cells(e.g., leukemia).

In another embodiment, therapeutic or pharmaceutical compositions asdescribed herein are administered to an animal to treat, prevent orameliorate growth, progression, and/or metastases of malignancies andproliferative diseases and disorders associated with increased cellsurvival, or the inhibition of apoptosis. In a specific embodiment, thepresent invention provides a method of treating a proliferative diseaseor disorder, comprising administering to an animal in which suchtreatment is desired, a BLyS binding polypeptide in an amount effectiveto treat the proliferative disease or disorder. For a review of suchdisorders, see Fishman et al., Medicine, 2d Ed. (J. B. Lippincott Co.,Philadelphia 1985). Examples of such disorders, include, but are notlimited to, leukemia (e.g., acute leukemia such as acute lymphocyticleukemia and acute myelocytic leukemia, myeloblastic leukemia,promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia,erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic)leukemia, and chronic lymphocytic leukemia), Polycythemia vera,lymphomas (e.g. Hodgkin's lymphoma, non-Hodgkin's lymphoma) Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, neoplasms, tumors (e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,nasopharyngeal carcinoma, bronchogenic carcinoma, esophageal carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma) heavy chain disease,metastases, or any disease or disorder characterized by uncontrolledcell growth. This method of treating a proliferative diseases ordisorders can also be used to treat premalignant conditions (e.g.,benign tumors, hyperproliferative disorders, and benign proliferativedisorders—see below) as well as proliferative disorders of B cells,monocytes, macrophages, and T cells.

In another embodiment of the present invention, therapeutic orpharmaceutical compositions as described herein can also be administeredto treat a subset of proliferative disorders, namely, premalignantconditions (e.g., benign tumors, hyperproliferative disorders, benignproliferative disorders) and to prevent progression to a neoplastic ormalignant state, including but not limited to those disorders listedabove. Such prophylactic or therapeutic use is indicated in conditionsknown or suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed. (W.B. Saunders Co., Philadelphia 1976), pp. 68-79.)Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a therapeutic orpharmaceutical composition as described herein. Characteristics of atransformed phenotype include, but are nor limited to, morphologychanges, looser substratum attachment, loss of contact inhibition, lossof anchorage dependence, protease release, increased sugar transport,decreased serum requirement, expression of fetal antigens, anddisappearance of the 250,000 dalton cell surface protein.

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a therapeutic or pharmaceuticalcomposition as described herein: a chromosomal translocation associatedwith a malignancy (e.g., the Philadelphia chromosome for chronicmyelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familialpolyposis or Gardner's syndrome (possible forerunners of colon cancer),benign monoclonal gammopathy (a possible forerunner of multiplemyeloma), and a first degree kinship with persons having a cancer orprecancerous disease showing a Mendelian (genetic) inheritance pattern(e.g., familial polyposis of the colon, Gardner's syndrome, hereditaryexostosis, polyendocrine adenomatosis, medullary thyroid carcinoma withamyloid production and pheochromocytoma, Peutz-Jeghers syndrome,neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid bodytumor, cutaneous melanocarcinoma, intraocular melanocarcinoma, xerodermapigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism,Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell,supra, pp. 112-113), etc.)

In a specific embodiment, therapeutic or pharmaceutical compositions asdescribed herein are used to treat or prevent a disorder characterizedby hypergammagloulinemia (e.g., AIDS, autoimmune diseases, and someimmunodeficiencies).

In a specific embodiment, therapeutic or pharmaceutical compositions asdescribed herein are used to treat or prevent a disorder characterizedby deficient serum immunoglobulin production, recurrent infections,and/or immune system dysfunction. Moreover, therapeutic orpharmaceutical compositions as described herein may be used to treat orprevent infections of the joints, bones, skin, and/or parotid glands,blood-borne infections (e.g., sepsis, meningitis, septic arthritis,and/or osteomyelitis), autoimmune diseases (e.g., those disclosedherein), inflammatory disorders, and malignancies, and/or any disease ordisorder or condition associated with these infections, diseases,disorders and/or malignancies) including, but not limited to, CVID,other primary immune deficiencies, HIV disease, CLL, recurrentbronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),and/or pheumocystis carnii.

Therapeutic or pharmaceutical compositions as described herein thereof,may be used to diagnose, prognose, treat or prevent one or more of thefollowing diseases or disorders, or conditions associated therewith:primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasakisyndrome, bone marrow transplant (e.g., recent bone marrow transplant inadults or children), chronic B-cell lymphocytic leukemia, HIV infection(e.g., adult or pediatric HIV infection), chronic inflammatorydemyelinating polyneuropathy, and post-transfusion purpura.

Additionally, therapeutic or pharmaceutical compositions as describedherein may be used to diagnose, prognose, treat or prevent one or moreof the following diseases, disorders, or conditions associatedtherewith, Guillain-Barre syndrome, anemia (e.g., anemia associated withparvovirus B19, patients with stable mutliple myeloma who are at highrisk for infection (e.g., recurrent infection), autoimmune hemolyticanemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia(e.g., neonatal thrombocytopenia), and immune-mediated neutropenia),transplantation (e.g., cytamegalovirus (CMV)-negative recipients ofCMV-positive organs), hypogammaglobulinemia (e.g.,hypogamma-globulinemic neonates with risk factor for infection ormorbidity), epilepsy (e.g., intractable epilepsy), systemic vasculiticsyndromes, myasthenia gravis (e.g., decompensation in myastheniagravis), dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are notlimited to, the use of therapeutic or pharmaceutical compositions asdescribed herein in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody production(e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.In a specific nonexclusive embodiment, therapeutic or pharmaceuticalcompositions as described herein are administered to boost the immunesystem to produce increased quantities of IgG. In another specificnonexclusive embodiment, BLyS binding polypeptides of the areadministered to boost the immune system to produce increased quantitiesof IgA. In another specific non-limiting embodiment, BLyS bindingpolypeptides are administered to boost the immune system to produceincreased quantities of IgM.

Administration to an animal (including, but not limited to, those listedabove, and also including transgenic animals) incapable of producingfunctional endogenous antibody molecules or having an otherwisecompromised endogenous immune system, but which is capable of producinghuman immunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT applications WO 98/24893, WO 96/34096, WO 96/33735, and WO91/10741).

Additional preferred embodiments of the invention include, but are notlimited to, the use of therapeutic or pharmaceutical compositions asdescribed herein in the following applications:

A vaccine adjuvant that enhances immune responsiveness to specificantigen. In a specific embodiment, the vaccine is a BLyS bindingpolypeptide described herein. In another specific embodiment, thevaccine adjuvant is a polynucleotide described herein (e.g., a BLySbinding polypeptide polynucleotide genetic vaccine adjuvant). Asdiscussed herein, therapeutic or pharmaceutical compositions asdescribed herein may be administered using techniques known in the art,including but not limited to, liposomal delivery, recombinant vectordelivery, injection of naked DNA, and gene gun delivery.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions as describedherein as an adjuvant, include, but are not limited to, virus and virusassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions are used as anadjuvant to enhance an immune response to a virus, disease, or symptomselected from the group consisting of: AIDS, meningitis, Dengue, EBV,and hepatitis (e.g., hepatitis B). In another specific embodiment, thecompositions are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting of:HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese Bencephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex, and yellow fever. In another specific embodiment, thecompositions as described herein are used as an adjuvant to enhance animmune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses.Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions as described herein as an adjuvant, includebacteria or fungus and bacteria or fungus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions as described herein are used as anadjuvant to enhance an immune response to a bacteria or fungus, disease,or symptom selected from the group consisting of: tetanus, diphtheria,botulism, and meningitis type B. In another specific embodiment, thecompositions are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonella paratyphi, Neisseria meningitidis, Streptococcus pneumoniae,Group B Streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasiticimmune responses that may be enhanced using the compositions asdescribed herein as an adjuvant, include parasite and parasiteassociated diseases or symptoms described herein or otherwise known inthe art. In specific embodiments, the compositions are used as anadjuvant to enhance an immune response to a parasite. In anotherspecific embodiment, the compositions are used as an adjuvant to enhancean immune response to Plasmodium (malaria).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogeneic or xenogeneicorgan transplantation). With respect to transplantation, compositions asdescribed herein may be administered prior to, concomitant with, and/orafter transplantation. In a specific embodiment, compositions areadministered after transplantation, prior to the beginning of recoveryof T cell populations. In another specific embodiment, compositions arefirst administered after transplantation, after the beginning ofrecovery of T cell populations, but prior to full recovery of B cellpopulations.

As an agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy. B cell immunodeficiencies that may beameliorated or treated by administering the BLyS binding polypeptidesand/or compositions as described herein include, but are not limited to,severe combined immunodeficiency (SCID)-X linked, SCID-autosomal,adenosine deaminase deficiency (ADA deficiency), X-linkedagammaglobulinemia (XLA), Bruton's disease, congenitalagammaglobulinemia, X-linked infantile agammaglobulinemia, acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non-X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

In a specific embodiment, BLyS binding polypeptides and/or compositionsare administered to treat or ameliorate selective IgA deficiency.

In another specific embodiment, BLyS binding polypeptides and/orcompositions are administered to treat or ameliorateataxia-telangiectasia.

In another specific embodiment BLyS binding polypeptides and/orcompositions are administered to treat or ameliorate common variableimmunodeficiency.

In another specific embodiment, BLyS binding polypeptides and/orcompositions are administered to treat or ameliorate X-linkedagammaglobulinemia.

In another specific embodiment, BLyS binding polypeptides and/orcompositions are administered to treat or ameliorate severe combinedimmunodeficiency (SCID).

In another specific embodiment, BLyS binding polypeptides and/orcompositions are administered to treat or ameliorate Wiskott-Aldrichsyndrome.

In another specific embodiment, BLyS binding polypeptides and/orcompositions are administered to treat or ameliorate X-linked Igdeficiency with hyper IgM.

As an agent to boost immunoresponsiveness among individuals having anacquired loss of B cell function. Conditions resulting in an acquiredloss of B cell function that may be ameliorated or treated byadministering BLyS binding polypeptides and/or compositions include, butare not limited to, HIV Infection, AIDS, bone marrow transplant, and Bcell chronic lymphocytic leukemia (CLL).

As an agent to boost immunoresponsiveness among individuals having atemporary immune deficiency. Conditions resulting in a temporary immunedeficiency that may be ameliorated or treated by administering BLySbinding polypeptides and/or compositions include, but are not limitedto, recovery from viral infections (e.g., influenza), conditionsassociated with malnutrition, recovery from infectious mononucleosis, orconditions associated with stress, recovery from measles, recovery fromblood transfusion, recovery from surgery.

As a regulator of antigen presentation by monocytes, dendritic cells, Tcells and/or B cells. In one embodiment, BLyS binding polypeptides orpolynucleotides enhance antigen presentation or antagonize antigenpresentation in vitro or in vivo. Moreover, in related embodiments, thisenhancement or antagonization of antigen presentation may be useful inanti-tumor treatment or to modulate the immune system.

As a mediator of mucosal immune responses. The expression of BLyS onmonocytes, the expression of BLyS receptor on B cells, and theresponsiveness of B cells to BLyS suggests that it may be involved inexchange of signals between B cells and monocytes or theirdifferentiated progeny. This activity is in many ways analogous to theCD40-CD 154 signaling between B cells and T cells. BLyS bindingpolypeptides and compositions may therefore be good regulators of T cellindependent immune responses to environmental pathogens. In particular,the unconventional B cell populations (CD5+) that are associated withmucosal sites and responsible for much of the innate immunity in humansmay respond to BLyS binding polypeptides or compositions as describedherein thereby enhancing or inhibiting individual's immune status.

As an agent to direct an individual's immune system towards developmentof a humoral response (i.e., TH2) as opposed to a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly, their susceptibility profile would likely change.

As a monocyte cell specific binding protein to which specific activatorsor inhibitors of cell growth may be attached. The result would be tofocus the activity of such activators or inhibitors onto normal,diseased, or neoplastic moncytic cell populations.

As a macrophage cell specific binding protein to which specificactivators or inhibitors of cell growth may be attached. The resultwould be to focus the activity of such activators or inhibitors ontonormal, diseased, or neoplastic macrophage cell populations.

As a B cell specific binding protein to which specific activators orinhibitors of cell growth may be attached. The result would be to focusthe activity of such activators or inhibitors onto normal, diseased, orneoplastic B cell populations.

As a means of detecting monocytic cells by virtue of its specificity.This application may require labeling the protein with biotin or otheragents (e.g., as described herein) to afford a means of detection.

As a means of detecting macrophage cells by virtue of its specificity.This application may require labeling the protein with biotin or otheragents (e.g., as described herein) to afford a means of detection.

As a means of detecting B-lineage cells by virtue of its specificity.This application may require labeling the protein with biotin or otheragents (e.g., as described herein) to afford a means of detection.

As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodificiency.

As part of a monocyte selection device the function of which is toisolate monocytes from a heterogenous mixture of cell types. BLySbinding polypeptides could be coupled to a solid support to whichmonocytes would then specifically bind. Unbound cells would be washedout and the bound cells subsequently eluted. A non-limiting use of thisselection would be to allow purging of tumor cells from, for example,bone marrow or peripheral blood prior to transplant.

As part of a B cell selection device the function of which is to isolateB cells from a heterogenous mixture of cell types. BLyS bindingpolypeptides (that do not inhibit BLys/BLys Receptor intereaction)binding soluble BLyS could be coupled to a solid support to which Bcells would then specifically bind. Unbound cells would be washed outand the bound cells subsequently eluted. A non-limiting use of thisselection would be to allow purging of tumor cells from, for example,bone marrow or peripheral blood prior to transplant.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceBLyS mediated responses.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecovery.

As a means of regulating secreted cytokines that are elicited by BLySand/or BLyS receptor.

BLyS binding polypeptides or polynucleotides may be used to modulate IgEconcentrations in vitro or in vivo.

Additionally, BLyS binding polypeptides or polynucleotides may be usedto treat, prevent, and/or diagnose IgE-mediated allergic reactions. Suchallergic reactions include, but are not limited to, asthma, rhinitis,and eczema.

In a specific embodiment, BLyS binding polypeptides or polynucleotidesare administered to treat, prevent, diagnose, and/or ameliorateselective IgA deficiency.

In another specific embodiment BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate ataxia-telangiectasia.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate common variable immunodeficiency.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate X-linked agammaglobulinemia.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate X-linked Ig deficiency with hyper IgM. In a specificembodiment BLyS binding polypeptides or polynucleotides are administeredto treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiencywith hyper IgM.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, and/or diagnosechronic myelogenous leukemia, acute myelogenous leukemia, leukemia,hystiocytic leukemia, monocytic leukemia (e.g., acute monocyticleukemia), leukemic reticulosis, Shilling Type monocytic leukemia,and/or other leukemias derived from monocytes and/or monocytic cellsand/or tissues.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate monocytic leukemoid reaction, as seen, for example, withtuberculosis.

In another specific embodiment, BLyS binding polypeptides orpolynucleotides are administered to treat, prevent, diagnose, and/orameliorate monocytic leukocytosis, monocytic leukopenia, monocytopenia,and/or monocytosis.

In a specific embodiment, BLyS binding polypeptides or polynucleotidesare used to treat, prevent, detect, and/or diagnose monocyte disordersand/or diseases, and/or conditions associated therewith.

In a specific embodiment, BLyS binding polypeptides or polynucleotidesare used to treat, prevent, detect, and/or diagnose primary B lymphocytedisorders and/or diseases, and/or conditions associated therewith. Inone embodiment, such primary B lymphocyte disorders, diseases, and/orconditions are characterized by a complete or partial loss of humoralimmunity. Primary B lymphocyte disorders, diseases, and/or conditionsassociated therewith that are characterized by a complete or partialloss of humoral immunity and that may be prevented, treated, detectedand/or diagnosed with compositions as described herein include, but arenot limited to, X-Linked Agammaglobulinemia (XLA), severe combinedimmunodeficiency disease (SCID), and selective IgA deficiency.

In a preferred embodiment BLyS binding polypeptides or polynucleotidesare used to treat, prevent, and/or diagnose diseases or disordersaffecting or conditions associated with any one or more of the variousmucous membranes of the body. Such diseases or disorders include, butare not limited to, for example, mucositis, mucoclasis, mucocolitis,mucocutaneous leishmaniasis (such as, for example, Americanleishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis,and New World leishmaniasis), mucocutaneous lymph node syndrome (forexample, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma,mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma,mucoid degeneration, myxoid degeneration; myxomatous degeneration;myxomatosis, mucoid medial degeneration (for example, cystic medialnecrosis), mucolipidosis (including, for example, mucolipidosis I,mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysisdisorders, mucomembranous enteritis, mucoenteritis,mucopolysaccharidosis (such as, for example, type Imucopolysaccharidosis (i.e., Hurler's syndrome), type ISmucopolysaccharidosis (i.e., Scheie's syndrome or type Vmucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter'ssyndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome),type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VImucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VIImucopolysaccharidosis (i.e, mucopolysaccharidosis due tobeta-glucuronidase deficiency), and mucosulfatidosis),mucopolysacchariduria, mucopurulent conjunctivitis, mucopus,mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virusdiarrhea), mucous colitis (such as, for example, mucocolitis andmyxomembranous colitis), and mucoviscidosis (such as, for example,cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfieldsyndrome, fibrocystic disease of the pancreas, mucoviscidosis, andviscidosis). In a highly preferred embodiment, BLyS binding polypeptidesor polynucleotides are used to treat, prevent, and/or diagnosemucositis, especially as associated with chemotherapy.

In a preferred embodiment, BLyS binding polypeptides or polynucleotidesare used to treat, prevent, and/or diagnose diseases or disordersaffecting or conditions associated with sinusitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by BLyS binding polypeptides orpolynucleotides is osteomyelitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by BLyS binding polypeptides orpolynucleotides is endocarditis.

All of the above described applications as they may apply to veterinarymedicine.

BLyS binding polypeptides or polynucleotides may be used to treat,prevent, and/or diagnose diseases and disorders of the pulmonary system(e.g., sinopulmonary and bronchial infections) and conditions associatedwith such diseases and disorders and other respiratory diseases anddisorders. In specific embodiments, such diseases and disorders include,but are not limited to, bronchial adenoma, bronchial asthma, pneumonia(such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculousbronchopneumonia), chronic obstructive pulmonary disease (COPD),bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca,cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolaradenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g.,exudative bronchiolitis, bronchiolitis fibrosa obliterans, andproliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchiticasthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani'sbronchitis, chronic bronchitis, croupous bronchitis, fibrinousbronchitis, hemorrhagic bronchitis, infectious avian bronchitis,obliterative bronchitis, plastic bronchitis, pseudomembranousbronchitis, putrid bronchitis, and verminous bronchitis), bronchocentricgranulomatosis, bronchoedema, bronchoesophageal fistula, bronchogeniccarcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia,bronchomycosis (such as, e.g., bronchopulmonary aspergillosis),bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea,bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration,bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration,respiratory acidosis, respiratory alkalosis, respiratory distresssyndrome of the newborn, respiratory insufficiency, respiratoryscleroma, respiratory syncytial virus, and the like.

In a specific embodiment, BLyS binding polypeptides or polynucleotidesare used to treat, prevent, and/or diagnose chronic obstructivepulmonary disease (COPD).

In another embodiment, BLyS binding polypeptides or polynucleotides areused to treat, prevent, and/or diagnose fibroses and conditionsassociated with fibroses, including, but not limited to, cystic fibrosis(including such fibroses as cystic fibrosis of the pancreas,Clarke-Hadfield syndrome, fibrocystic disease of the pancreas,mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathicretroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis,nodular subepidermal fibrosis, pericentral fibrosis, perimuscularfibrosis, pipestem fibrosis, replacement fibrosis, subadventitialfibrosis, and Symmers' clay pipestem fibrosis.

In another embodiment, therapeutic or pharmaceutical compositions areadministered to an animal to treat, prevent or ameliorate infectiousdiseases. Infectious diseases include diseases associated with yeast,fungal, viral and bacterial infections. Viruses causing viral infectionswhich can be treated or prevented in accordance with this inventioninclude, but are not limited to, retroviruses (e.g., human T-celllymphotrophic virus (HTLV) types I and II and human immunodeficiencyvirus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types Iand II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenavirues(e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus virus,human respiratory syncytial virus, mumps, and pneumovirus),adenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses(e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellowfever virus, and Japanese encephalitis virus), hepadnaviruses (e.g.,hepatitis B viruses (HBV)), orthomyoviruses (e.g., influenza viruses A,B and C), papovaviruses (e.g., papillomavirues), picornaviruses (e.g.,rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses,reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus),rhabdoviruses (e.g., rabies virus). Microbial pathogens causingbacterial infections include, but are not limited to, Streptococcuspyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseriameningitidis, Corynebacterium diphtheriae, Clostridium botulinum,Clostridium perfringens, Clostridium tetani, Haemophilus influenzae,Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis,Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonasaeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni,Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium,Treponema pallidum, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii,Francisella tularensis, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp., and Helicobacter pylori.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingBLyS binding polypeptides or functional derivatives thereof, areadministered to treat, inhibit or prevent a disease or disorderassociated with aberrant expression and/or activity of BLyS and/or itsreceptor, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded protein that mediates a therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy, 12:488-505 (1993); Wu and Wu, Biotherapy,3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596(1993); Mulligan, Science, 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem., 62:191-217 (1993); May, TIBTECH, 11(5):155-215(1993). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Current Protocols in MolecularBiology, Ausubel et al., eds. (John Wiley & Sons, NY 1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual (StocktonPress, NY 1990).

In a preferred aspect, a composition useful in the methods of theinvention comprises, or alternatively consists of, nucleic acidsencoding a BLyS binding polypeptide, said nucleic acids being part of anexpression vector that expresses the BLyS binding polypeptide orfragment thereof or chimeric protein including it in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the BLyS binding polypeptide codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the BLyS binding polypeptide codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the BLyS bindingpolypeptide encoding nucleic acids (Koller and Smithies, Proc. Natl.Acad. Sci. USA, 86:8932-8935 (1989); Zijlstra et al., Nature,342:435-438 (1989).

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA, 86:8932-8935 (1989); Zijlstra et al., Nature,342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding a BLyS binding polypeptide or fragments or variantsthereof are used. For example, a retroviral vector can be used (seeMiller et al., Meth. Enzmmol., 217:581-599 (1993)). These retroviralvectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA. The nucleicacid sequences encoding the BLyS binding polypeptide to be used in genetherapy are cloned into one or more vectors, which facilitates deliveryof the gene into a patient. Additional details concerning retroviralvectors can be found in Boesen et al., Biotherapy, 6:29 1-302 (1994),which describes the use of a retroviral vector to deliver the mdr 1 geneto hematopoietic stem cells in order to make the stem cells moreresistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.,93:644-651(1994); Klein et al., Blood, 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy, 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel., 3:110-114 (1993).

Other viral vectors that can be used in gene therapy are adenoviruses.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia, where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. See, Kozarsky and Wilson,Current Opinion in Genetics and Development, 3:499-503 (1993),presenting a review of adenovirus-based gene therapy. Bout et al., HumanGene Therapy, 5:3-10 (1994) demonstrated the use of adenovirus vectorsto transfer genes to the respiratory epithelia of rhesus monkeys. Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell,68:143-155 (1992); Mastrangeli et al., J. Clin. Invest., 91:225-234(1993); PCT publication WO 94/12649; and Wang et al., Gene Therapy,2:775-783 (1995). In a preferred embodiment, adenovirus vectors areused.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med., 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.,217:599-618 (1993); Cohen et al., Meth. Enzymol., 217:618-644 (1993);Clin. Pharma. Ther., 29:69-92m (1985)) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding a BLyS binding polypeptide or fragmentthereof are introduced into the cells such that they are expressible bythe cells or their progeny, and the recombinant cells are thenadministered in vivo for therapeutic effect. In a specific embodiment,stem or progenitor cells are used. Any stem and/or progenitor cells thatcan be isolated and maintained in vitro can potentially be used inaccordance with this embodiment of the present invention (see, e.g., PCTpublication WO 94/08598; Stemple and Anderson, Cell, 7 1:973-985 (1992);Rheinwald, Meth. Cell Bio., 21A:229 (1980); and Pittelkow and Scott,Mayo Clinic Proc., 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Utility of a Composition

The compounds are preferably tested in vitro, and then in vivo for thedesired therapeutic or prophylactic activity, prior to use in humans.For example, in vitro assays which can be used to determine whetheradministration of a specific BLyS binding polypeptide or composition ofthe present invention is indicated, include in vitro cell culture assaysin which a patient tissue sample is grown in culture, and exposed to, orotherwise administered, a BLyS binding polypeptide or composition of thepresent invention, and the effect of such a BLyS binding polypeptide orcomposition of the present invention upon the tissue sample is observed.In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a BLyS binding polypeptide or composition of the presentinvention has a desired effect upon such cell types. Preferably, theBLyS binding polypeptides or compositions are also tested in in vitroassays and animal model systems prior to administration to humans.

BLyS binding polypeptides or compositions of the present invention foruse in therapy can be tested for their toxicity in suitable animal modelsystems, including but not limited to rats, mice, chicken, cows,monkeys, and rabbits. For in vivo testing of a BLyS binding polypeptideor composition's toxicity any animal model system known in the art maybe used.

Efficacy in treating or preventing viral infection may be demonstratedby detecting the ability of a BLyS binding polypeptide or composition toinhibit the replication of the virus, to inhibit transmission or preventthe virus from establishing itself in its host, or to prevent,ameliorate or alleviate the symptoms of disease a progression. Thetreatment is considered therapeutic if there is, for example, areduction in viral load, amelioration of one or more symptoms, or adecrease in mortality and/or morbidity following administration of aBLyS binding polypeptide or composition.

BLyS binding polypeptides or compositions can be tested for the abilityto induce the expression of cytokines such as IFN-γ, by contactingcells, preferably human cells, with a BLyS binding polypeptide orcomposition or a control BLyS binding polypeptide or control compositionand determining the ability of the BLyS binding polypeptide orcomposition to induce one or more cytokines. Techniques known to thoseskilled in the art can be used to measure the level of expression ofcytokines. For example, the level of expression of cytokines can bemeasured by analyzing the level of RNA of cytokines by, for example,RT-PCR and Northern blot analysis, and by analyzing the level ofcytokines by, for example, immunoprecipitation followed by western blotanalysis and ELISA. In a preferred embodiment, a compound is tested forits ability to induce the expression of IFN-γ.

BLyS binding polypeptides or compositions can be tested for theirability to modulate the biological activity of immune cells bycontacting immune cells, preferably human immune cells (e.g., T cells, Bcells, and Natural Killer cells), with a BLyS binding polypeptide orcomposition or a control compound and determining the ability of theBLyS binding polypeptide or compostion to modulate (i.e, increase ordecrease) the biological activity of immune cells. The ability of a BLySbinding polypeptide or composition to modulate the biological activityof immune cells can be assessed by detecting the expression of antigens,detecting the proliferation of immune cells (i.e., B cellproliferation), detecting the activation of signaling molecules,detecting the effector function of immune cells, or detecting thedifferentiation of immune cells. Techniques known to those of skill inthe art can be used for measuring these activities. For example,cellular proliferation can be assayed by ³H-thymidine incorporationassays and trypan blue cell counts. Antigen expression can be assayed,for example, by immunoassays including, but not limited to, competitiveand non-competitive assay systems using techniques such as westernblots, immunohistochemistry radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays and FACS analysis. The activation of signaling moleculescan be assayed, for example, by kinase assays and electrophoretic shiftassays (EMSAs). In a preferred embodiment, the ability of a BLyS bindingpolypeptide or composition to induce B cell proliferation is measured.In another preferred embodiment, the ability of a BLyS bindingpolypeptide or composition to modulate immunoglobulin expression ismeasured.

BLyS binding polypeptides or compositions can be tested for theirability to reduce tumor formation in in vitro, ex vivo and in vivoassays. BLyS binding polypeptides or compositions can also be tested fortheir ability to inhibit viral replication or reduce viral load in invitro and in vivo assays. BLyS binding polypeptides or compositions canalso be tested for their ability to reduce bacterial numbers in in vitroand in vivo assays known to those of skill in the art. BLyS bindingpolypeptides or compositions can also be tested for their ability toalleviate of one or more symptoms associated with cancer, an immunedisorder (e.g., an inflammatory disease), a neurological disorder or aninfectious disease. BLyS binding polypeptides or compositions can alsobe tested for their ability to decrease the time course of theinfectious disease. Further, BLyS binding polypeptides or compositionscan be tested for their ability to increase the survival period ofanimals suffering from disease or disorder, including cancer, an immunedisorder or an infectious disease. Techniques known to those of skill inthe art can be used to analyze the function of the BLyS bindingpolypeptides or compositions in vivo.

Therapeutic/Prophylactic Compositions and Administration

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of BLyS bindingpolypeptide (or fragment or variant thereof) or pharmaceuticalcomposition, preferably a BLyS binding polypeptide. In a preferredaspect, a BLyS binding polypeptide or fragment or variant thereof issubstantially purified (i.e., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to, animals such ascows, pigs, horses, chickens, cats, dogs, etc., and is preferably amammal, and most preferably a human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer BLySbinding polypeptide or fragment or variant thereof, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the BLyS binding polypeptide or BLyS binding polypeptidefragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol.Chem., 262:4429-4432 (1987)), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include, butare not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. Preferably, whenadministering a protein, including a BLyS binding polypeptide, care mustbe taken to use materials to which the protein does not absorb.

In another embodiment, the composition can be delivered in a vesicle, inparticular a liposome (see, Langer, Science, 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see, generally, ibid.).

In yet another embodiment, the composition can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987);Buchwald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl. J.Med., 321:574 (1989)). In another embodiment, polymeric materials can beused (see, Medical Applications of Controlled Release, Langer and Wise,eds. (CRC Press, Boca Raton, Fla. 1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball,eds. (Wiley, New York 1984); Ranger and Peppas, Macromol. Sci. Rev.Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190(1985); During et al., Ann. Neurol., 25:35 1 (1989); Howard et al., J.Neurosurg., 7 1:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,e.g., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (Science, 249:1527-1533 (1990)).

In a specific embodiment where the composition to be used in the methodof the invention is a nucleic acid encoding a protein, the nucleic acidcan be administered in vivo to promote expression of its encodedprotein, by constructing it as part of an appropriate nucleic acidexpression vector and administering it so that it becomes intracellular,e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or bydirect injection, or by use of microparticle bombardment (e.g., a genegun; Biolistic, Dupont), or coating with lipids or cell-surfacereceptors or transfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see, e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-1868 (1991)), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a BLySbinding polypeptide or a fragment thereof, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations, andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed. (Mack Publishing Co., 1990). Such compositions will contain atherapeutically effective amount of the BLyS binding polypeptide orfragment thereof, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions for use in the methods of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylaminoethanol, histidine, procaine, etc.

The amount of the composition which will be effective in the treatment,inhibition and prevention of a disease or disorder associated withaberrant expression and/or activity of a polypeptide can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For BLyS binding polypeptides, the dosage administered to a patient istypically 0.1 mg/kg to 100 mg/kg of the patient's body weight.Preferably, the dosage administered to a patient is between 0.1 mg/kgand 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10mg/kg of the patient's body weight. Further, the dosage and frequency ofadministration of therapeutic or pharmaceutical compositions may bereduced by enhancing uptake and tissue penetration (e.g., into thebrain) of the BLyS binding polypeptides by modifications such as, forexample, lipidation.

The BLyS binding polypeptides and BLyS binding polypeptide compositionsmay be administered alone or in combination with other moleculesincluding BLyS. In further embodiments of the invention, the BLySbinding polypeptides are administered in complex with BLyS. Preferablythe BLyS binding polypeptide is radiolabelled or in complex with aradioisotope, toxin, or prodrug. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The BLyS binding polypeptides and BLyS binding polypeptide compositionsmay be administered alone or in combination with other adjuvants.Adjuvants that may be administered with the BLyS binding polypeptidesand BLyS binding polypeptide compositions include, but are not limitedto, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.),QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, BLySbinding polypeptides and BLyS binding polypeptide compositions areadministered in combination with alum. In another specific embodiment,BLyS binding polypeptides and BLyS binding polypeptide compositions areadministered in combination with QS-21. Further adjuvants that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, Monophosphoryllipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminumsalts, MF-59, and Virosomal adjuvant technology. Vaccines that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, vaccinesdirected toward protection against MMR (measles, mumps, rubella), polio,varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilusinfluenzae B, whooping cough, pneumonia, influenza, Lyme's Disease,rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis,rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23™.

In another specific embodiment, BLyS binding polypeptides and BLySbinding polypeptide compositions are used in combination withPNEUMOVAX-23™ to treat, prevent, and/or diagnose infection and/or anydisease, disorder, and/or condition associated therewith. In oneembodiment, BLyS binding polypeptides and BLyS binding polypeptidecompositions are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose any Gram positive bacterial infection and/orany disease, disorder, and/or condition associated therewith. In anotherembodiment, BLyS binding polypeptides and BLyS binding polypeptidecompositions are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the genus Enterococcusand/or the genus Streptococcus. In another embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions are used in anycombination with PNEUMOVAX-23™ to treat, prevent, and/or diagnoseinfection and/or any disease, disorder, and/or condition associated withone or more members of the Group B streptococci. In another embodiment,BLyS binding polypeptides and BLyS binding polypeptide compositions areused in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with Streptococcus pneumoniae.

The BLyS binding polypeptides and BLyS binding polypeptide compositionsmay be administered alone or in combination with other therapeuticagents, including but not limited to, chemotherapeutic agents,antibiotics, antivirals, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents andcytokines. Combinations may be administered either concomitantly, e.g.,as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with othermembers of the TNF family. TNF, TNF-related or TNF-like molecules thatmay be administered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, soluble formsof TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta),LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL,CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (PCT publication WO96/14328), TRAIL, AIM-II (PCT publication WO 97/34911), APRIL (J. Exp.Med., 188(6):1185-1190 (1998)), endokine-alpha (PCT publication WO98/07880), Neutrokine-alpha (PCT publication WO 98/18921), OPG, OX40,and nerve growth factor (NGF), and soluble forms of fas, CD30, CD27,CD40 and 4-IBB, TR2 (PCT publication WO 96/34095), DR3 (PCT publicationWO 97/33904), DR4 (PCT publication WO 98/32856), TR5 (PCT publication WO98/30693), TR6 (PCT publication WO 98/30694), TR7 (PCT publication WO98/41629), TRANK, TR9 (PCT publication WO 98/56892), 312C2 (PCTpublication WO 98/06842), and TR12, and soluble forms CD154, CD70, andCD153.

In a preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withCD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™),bioloigically active fragments, variants, or derivatives of CD40L,anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),and/or anti-CD40 antibodies (e.g., agonistic or antagonisticantibodies).

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered alone or incombination with an anti-angiogenic agent(s). Anti-angiogenic agentsthat may be administered with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston LifeSciences, Boston, Mass.), anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res., 51:22-26, 1991); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem., 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J., 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature,348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff,J. Clin. Invest., 79:1440-1446, 1987); anticollagenase-serum;alpha2-antiplasmin (Holmes et al., J. Biol. Chem., 262(4):1659-1664,1987); Bisantrene (National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions, 36:312-316, 1992); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (Brem and Folkman, J. Pediatr.Surg., 28:445-51 (1993)); an integrin alpha v beta 3 antagonist(Storgard et al., J. Clin. Invest., 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administered in combination with thecompounds may work through a variety of mechanisms including, but notlimited to, inhibiting proteolysis of the extracellular matrix, blockingthe function of endothelial cell-extracellular matrix adhesionmolecules, by antagonizing the function of angiogenesis inducers such asgrowth factors, and inhibiting integrin receptors expressed onproliferating endothelial cells. Examples of anti-angiogenic inhibitorsthat interfere with extracellular matrix proteolysis and which may beadministered in combination with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven,Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, LaJolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenicagents that act by directly antagonizing or inhibiting angiogenesisinducers and which may be administered in combination with the BLySbinding polypeptides and BLyS binding polypeptide compositions include,but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGFBLyS binding polypeptide (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.),and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectlyinhibit angiogenesis. Examples of indirect inhibitors of angiogenesiswhich may be administered in combination with the BLyS bindingpolypeptides and BLyS binding polypeptide compositions include, but arenot limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha,IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (GeorgetownUniversity, Washington, D.C.).

In particular embodiments, the use of BLyS binding polypeptides and BLySbinding polypeptide compositions in combination with anti-angiogenicagents is contemplated for the treatment, prevention, and/oramelioration of an autoimmune disease, such as for example, anautoimmune disease described herein.

In a particular embodiment, the use of BLyS binding polypeptides andBLyS binding polypeptide compositions in combination withanti-angiogenic agents is contemplated for the treatment, prevention,and/or amelioration of arthritis. In a more particular embodiment, theuse of BLyS binding polypeptides and BLyS binding polypeptidecompositions in combination with anti-angiogenic agents is contemplatedfor the treatment, prevention, and/or amelioration of rheumatoidarthritis.

In another embodiment, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with ananticoagulant. Anticoagulants that may be administered with the BLySbinding polypeptides and BLyS binding polypeptide compositions include,but are not limited to, heparin, warfarin, and aspirin. In a specificembodiment, BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with heparin and/orwarfarin. In another specific embodiment, BLyS binding polypeptides andBLyS binding polypeptide compositions are administered in combinationwith warfarin. In another specific embodiment, BLyS binding polypeptidesand BLyS binding polypeptide compositions are administered incombination with warfarin and aspirin. In another specific embodiment,BLyS binding polypeptides and BLyS binding polypeptide compositions areadministered in combination with heparin. In another specificembodiment, BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with heparin and aspirin.

In another embodiment, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with an agentthat suppresses the production of anticardiolipin antibodies. Inspecific embodiments, the polypeptides are administered in combinationwith an agent that blocks and/or reduces the ability of anticardiolipinantibodies to bind phospholipid-binding plasma protein beta2-glycoprotein I (b2GPI).

In certain embodiments, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination withanti-retroviral agents, nucleoside reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, and/or proteaseinhibitors. Nucleoside reverse transcriptase inhibitors that may beadministered in combination with the BLyS binding polypeptides and BLySbinding polypeptide compositions, include, but are not limited to,RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™(zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), andCOMBIVIR™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptaseinhibitors that may be administered in combination with the BLyS bindingpolypeptides and BLyS binding polypeptide compositions, include, but arenot limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), andSUSTIVA™ (efavirenz). Protease inhibitors that may be administered incombination with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions, include, but are not limited to, CRIXIVAN™(indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™(nelfinavir). In a specific embodiment, antiretroviral agents,nucleoside reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, and/or protease inhibitors may be used in anycombination with BLyS binding polypeptides and BLyS binding polypeptidecompositions to treat, prevent, and/or diagnose AIDS and/or to treat,prevent, and/or diagnose HIV infection.

In other embodiments, BLyS binding polypeptides and BLyS bindingpolypeptide compositions may be administered in combination withanti-opportunistic infection agents. Anti-opportunistic agents that maybe administered in combination with the BLyS binding polypeptides andBLyS binding polypeptide compositions, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICLOVIR™,PYRIMETHAMNE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions are used in anycombination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™,and/or ATOVAQUONE™ to prophylactically treat, prevent, and/or diagnosean opportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are used in any combination with ISONIAZID™,RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat,prevent, and/or diagnose an opportunistic Mycobacterium avium complexinfection. In another specific embodiment, BLyS binding polypeptides andBLyS binding polypeptide compositions are used in any combination withRIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions are used in anycombination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ toprophylactically treat, prevent, and/or diagnose an opportunisticcytomegalovirus infection. In another specific embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat, prevent, and/or diagnose an opportunistic fungalinfection. In another specific embodiment, BLyS binding polypeptides andBLyS binding polypeptide compositions are used in any combination withACYCLOVIR™ and/or FAMCICLOVIR™ to prophylactically treat, prevent,and/or diagnose an opportunistic herpes simplex virus type I and/or typeII infection. In another specific embodiment, BLyS binding polypeptidesand BLyS binding polypeptide compositions are used in any combinationwith PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat,prevent, and/or diagnose an opportunistic Toxoplasma gondii infection.In another specific embodiment, BLyS binding polypeptides and BLySbinding polypeptide compositions are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/ordiagnose an opportunistic bacterial infection.

In a further embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with anantiviral agent. Antiviral agents that may be administered with the BLySbinding polypeptides and BLyS binding polypeptide compositions include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with anantibiotic agent. Antibiotic agents that may be administered with theBLyS binding polypeptides and BLyS binding polypeptide compositionsinclude, but are not limited to, amoxicillin, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,steroids, cyclosporine, cyclosporine analogs cyclophosphamide,cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, BLyS binding polypeptides and BLYS bindingpolypeptide compositions are administered in combination withimmunosuppressants. Immunosuppressant preparations that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, ORTHOCLONE™(OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™(tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine,glucorticosteroids, and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

In a preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withsteroid therapy. Steroids that may be administered in combination withthe BLyS binding polypeptides and BLyS binding polypeptide compositions,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with prednisone. In afurther specific embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions and prednisone are those described herein, andinclude, but are not limited to, azathioprine, cylophosphamide, andcyclophosphamide IV. In a another specific embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions are administeredin combination with methylprednisolone. In a further specificembodiment, the BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with methylprednisolone andan immunosuppressive agent. Immunosuppressive agents that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions and methylprednisolone are those describedherein, and include, but are not limited to, azathioprine,cylophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination with anantimalarial. Antimalarials that may be administered with the BLySbinding polypeptides and BLyS binding polypeptide compositions include,but are not limited to, hydroxychloroquine, chloroquine, and/orquinacrine.

In a preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination with anNSAID.

In a nonexclusive embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withone, two, three, four, five, ten, or more of the following drugs:NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozinpotassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexeddisodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto),eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (CelltechChiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KSBiomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis),JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha),darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen;Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade(Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (BoehringerIngelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics),MK-663 (Merck), ST-1482 (Sigma-Tau), and butixocort propionate(WarnerLambert).

In a preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withone, two, three, four, five or more of the following drugs:methotrexate, sulfasalazine, sodium aurothiomalate, auranofin,cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g.,as described herein), cyclophosphamide, chlorambucil, gold, ENBREL™(Etanercept), anti-TNF antibody, LJP 394 (La Jolla PharmaceuticalCompany, San Diego, Calif.) and prednisolone.

In a more preferred embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination with anantimalarial, methotrexate, anti-TNF antibody, REMICADE™, ENBREL™ and/orsuflasalazine. In one embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withmethotrexate. In another embodiment, the BLyS binding polypeptides andBLyS binding polypeptide compositions are administered in combinationwith anti-TNF antibody. In another embodiment, the BLyS bindingpolypeptides and BLyS binding polypeptide compositions are administeredin combination with methotrexate and anti-TNF antibody. In anotherembodiment, the BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with suflasalazine. Inanother specific embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withmethotrexate, anti-TNF antibody, and suflasalazine. In anotherembodiment, the BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination ENBREL™. In anotherembodiment, the BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with ENBREL™ andmethotrexate. In another embodiment, the BLyS binding polypeptides andBLyS binding polypeptide compositions are administered in combinationwith ENBREL™, methotrexate and suflasalazine. In another embodiment, theBLyS binding polypeptides and BLyS binding polypeptide compositions areadministered in combination with ENBREL™, methotrexate andsuflasalazine. In other embodiments, one or more antimalarials iscombined with one of the above-recited combinations. In a specficembodiment, the BLyS binding polypeptides and BLyS binding polypeptidecompositions are administered in combination with an antimalarial (e.g.,hydroxychloroquine), ENBREL™, methotrexate and suflasalazine. In anotherspecfic embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with anantimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNFantibody, and methotrexate.

In an additional embodiment, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered alone or in combination withone or more intravenous immune globulin preparations. Intravenous immuneglobulin preparations that may be administered with the BLyS bindingpolypeptides and BLyS binding polypeptide compositions include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withintravenous immune globulin preparations in transplantation therapy(e.g., bone marrow transplant).

CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™),biologically active fragments, variants, or derivatives of CD40L,anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),and/or anti-CD40 antibodies (e.g., agonistic or antagonisticantibodies).

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered alone or incombination with an anti-inflammatory agent. Anti-inflammatory agentsthat may be administered with the BLyS binding polypeptides and BLySbinding polypeptide compositions include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

In another embodiment, compostions are administered in combination witha chemotherapeutic agent. Chemotherapeutic agents that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, antibioticderivatives (e.g., doxorubicin, bleomycin, daunorubicin, anddactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g.,fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b,glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxicagents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone) or anycombination of the components of CHOP. In another embodiment, BLySbinding polypeptides and BLyS binding polypeptide compositions areadministered in combination with Rituximab. In a further embodiment,BLyS binding polypeptides and BLyS binding polypeptide compositions areadministered with Rituxmab and CHOP, or Rituxmab and any combination ofthe components of CHOP.

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withcytokines. Cytokines that may be administered with the BLyS bindingpolypeptides and BLyS binding polypeptide compositions include, but arenot limited to, GM-CSF, G-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-10, IL-12, IL13, IL-15, anti-CD40, CD40L, IFN-alpha (IFN-α), IFN-beta(IFN-β), IFN-gamma (IFN-γ), TNF-alpha (TNF-α), and TNF-beta (TNF-β). Inpreferred embodiments, BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered with BLyS (e.g., amino acids134-285 of SEQ ID NO:173). In another embodiment, BLyS bindingpolypeptides and BLyS binding polypeptide compositions may beadministered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, and IL-22. In preferred embodiments, the BLyS bindingpolypeptides and BLyS binding polypeptide compositions are administeredin combination with IL-4 and IL-10.

In one embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered in combination with one ormore chemokines. In specific embodiments, the BLyS binding polypeptidesand BLyS binding polypeptide compositions are administered incombination with an α(CxC) chemokine selected from the group consistingof gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8),platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α,GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocytechemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1(SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a β(CC)chemokine selected from the group consisting of: RANTES (regulated onactivation, normal T expressed and secreted), macrophage inflammatoryprotein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta(MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotacticprotein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocytechemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma(MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophageinflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4(MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and 1-309; and/or the γ(C)chemokine, lymphotactin.

In another embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered with chemokine beta-8,chemokine beta-1, and/or macrophage inflammatory protein-4. In apreferred embodiment, the BLyS binding polypeptides and BLyS bindingpolypeptide compositions are administered with chemokine beta-8.

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination with anIL-4 antagonist. IL-4 antagonists that may be administered with the BLySbinding polypeptides and BLyS binding polypeptide compositions include,but are not limited to: soluble IL-4 receptor polypeptides, multimericforms of soluble IL-4 receptor polypeptides; anti-IL-4 receptorantibodies that bind the IL-4 receptor without transducing thebiological signal elicited by IL-4, anti-IL4 antibodies that blockbinding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 thatbind IL-4 receptors but do not transduce the biological signal elicitedby IL-4. Preferably, the antibodies employed according to this methodare monoclonal antibodies (including BLyS binding polypeptide fragments,such as, for example, those described herein).

The invention also encompasses combining the polynucleotides and/orpolypeptides (and/or agonists or antagonists thereof) with otherproposed or conventional hematopoietic therapies. Thus, for example, thepolynucleotides and/or polypeptides (and/or agonists or antagoniststhereof) can be combined with compounds that singly exhibiterythropoietic stimulatory effects, such as erythropoietin,testosterone, progenitor cell stimulators, insulin-like growth factor,prostaglandins, serotonin, cyclic AMP, prolactin, and triiodothyzonine.Also encompassed are combinations of the BLyS binding polypeptides andBLyS binding polypeptide compositions with compounds generally used totreat aplastic anemia, such as, for example, methenolene, stanozolol,and nandrolone; to treat iron-deficiency anemia, such as, for example,iron preparations; to treat malignant anemia, such as, for example,vitamin B₁₂ and/or folic acid; and to treat hemolytic anemia, such as,for example, adrenocortical steroids, e.g., corticoids. See, e.g.,Resegotti et al., Panminerva Medica, 23:243-248 (1981); Kurtz, FEBSLetters, 14a:105-108 (1982); McGonigle et al., Kidney Int., 25:437-444(1984); and Pavlovic-Kantera, Expt. Hematol., 8(supp. 8):283-291 (1980),the contents of each of which are hereby incorporated by reference intheir entireties.

Compounds that enhance the effects of or synergize with erythropoietinare also useful as adjuvants herein, and include but are not limited to,adrenergic agonists, thyroid hormones, androgens, hepatic erythropoieticfactors, erythrotropins, and erythrogenins. See, for example, Dunn,Current Concepts in Erythropoiesis (John Wiley and Sons, Chichester,England 1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, NewEng. J. Med., 289:72-80 (1973); Urabe et al., J. Exp. Med.,149:1314-1325 (1979); Billat et al., Expt. Hematol., 10:133-140 (1982);Naughton et al., Acta Haemat., 69:171-179 (1983); Cognote et al., inabstract 364, Proceedings 7th Intl. Cong. of Endocrinology (Quebec City,Quebec, Jul. 1-7, 1984); and Rothman et al., J. Surg. Oncol., 20:105-108(1982). Methods for stimulating hematopoiesis comprise administering ahematopoietically effective amount (i.e., an amount which effects theformation of blood cells) of a pharmaceutical composition containingpolynucleotides and/or poylpeptides (and/or agonists or antagoniststhereof) to a patient. The polynucleotides and/or polypeptides and/oragonists or antagonists thereof are administered to the patient by anysuitable technique, including but not limited to parenteral, sublingual,topical, intrapulmonary and intranasal, and those techniques furtherdiscussed herein. The pharmaceutical composition optionally contains oneor more members of the group consisting of erythropoietin, testosterone,progenitor cell stimulators, insulin-like growth factor, prostaglandins,serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene,stanozolol, and nandrolone, iron preparations, vitamin B₁₂, folic acidand/or adrenocortical steroids.

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withhematopoietic growth factors. Hematopoietic growth factors that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to,LEUKINE™(sargramostim) and NEUPOGEN™ (filgrastim).

In an additional embodiment, the BLyS binding polypeptides and BLySbinding polypeptide compositions are administered in combination withfibroblast growth factors. Fibroblast growth factors that may beadministered with the BLyS binding polypeptides and BLyS bindingpolypeptide compositions include, but are not limited to, FGF-1, FGF-2,FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

Additionally, the BLyS binding polypeptides and BLyS binding polypeptidecompositions may be administered alone or in combination with othertherapeutic regimens, including but not limited to, radiation therapy.Such combinatorial therapy may be administered sequentially and/orconcomitantly.

Kits for Detecting and/or Quantitating BLyS or BLyS-Like Polypeptides

The present invention is also directed to an assay kit which can beuseful in screening for the presence of BLyS and/or quantitating BLySconcentrations in a fluid, such as, for example, a biological fluid(e.g., blood, serum, synovial fluid).

In a particular embodiment of the present invention, an assay kit iscontemplated which comprises in one or more containers one or more BLySbinding polypeptides and optionally, a detection means for determiningthe presence of a BLyS-BLyS binding polypeptide interaction or theabsence thereof. The kit further optionally contains BLyS protein thatmay be used, for example as a control. The BLyS binding polypeptide maybe free or expressed on the surface of a phage.

In a specific embodiment, either the BLyS binding polypeptide or theBLyS protein is labeled. As further discussed herein, a wide range oflabels can be used accordance with the present invention, including butnot limited to conjugating the recognition unit to biotin byconventional means. Alternatively, the label may comprise, e.g., afluorogen, an enzyme, an epitope, a chromogen, or a radionuclide.Preferably, the biotin is conjugated by covalent attachment to eitherthe BLyS binding polypeptide or the BLyS protein. Preferably, the BLySbinding polypeptide is immobilized on a solid support. The detectionmeans employed to detect the label will depend on the nature of thelabel and can be any known in the art, e.g., film to detect aradionuclide, an enzyme substrate that gives rise to a detectable signalto detect the presence of an enzyme, antibody to detect the presence ofan epitope, etc.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In one preferredembodiment the kit comprises a vial containing BLyS binding polypeptidesconjugated to a toxin or a label (as described herein). Such conjugatedbinding polypeptide may be used to kill a particular population of cellsor to quantitate a particular population of cells. In a preferredembodiment, such conjugated BLyS binding polypeptides are used to killmonocyte cells expressing the membrane-bound form of BLyS. In anotherpreferred embodiment, such conjugated BLyS binding polypeptides are usedto quantitate monocyte cells expressing the membrane-bound form of BLyS.In another preferred embodiment, such conjugated BLyS bindingpolypeptides are used to kill B cells expressing BLyS receptor on theirsurface. In another preferred embodiment, such conjugated BLyS bindingpolypeptides are used to quantitate B cells expressing BLyS receptor ontheir surface.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises a BLyS binding polypeptide,preferably a purified BLyS binding polypeptide, in one or morecontainers. In an alterative embodiment, a kit comprises a BLyS bindingpolypeptide fragment that specifically binds to BLyS. In a specificembodiment, the kits of the present invention contain a substantiallyisolated BLyS polypeptide as a control. Preferably, the kits of thepresent invention further comprise a control binding polypeptide whichdoes not react with BLyS. In another specific embodiment, the kits ofthe present invention contain a means for detecting the binding of aBLyS binding polypeptide to BLyS (e.g., the BLyS binding polypeptide maybe conjugated to a detectable substrate such as a fluorescent compound,an enzymatic substrate, a radioactive compound or a luminescentcompound, or a second antibody which recognizes the BLyS bindingpolypeptide may be conjugated to a detectable substrate). In specificembodiments, the kit may include a recombinantly produced or chemicallysynthesized BLyS. The BLyS provided in the kit may also be attached to asolid support. In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which BLyS is attached.Such a kit may also include a non-attached reporter-labeled anti-BLySbinding polypeptide antibody. In this embodiment, binding of the BLySbinding polypeptide to BLyS can be detected by binding of the saidreporter-labeled antibody. Alternatively, or in addition, the detectingmeans may include a labeled, competing antigen.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing BLyS or BLyS-like polypeptides. Thediagnostic kit includes a substantially isolated BLyS bindingpolypeptide specifically reactive with BLyS target, and means fordetecting the binding of BLyS target to the BLyS binding polypeptide. Inone embodiment, the BLyS binding polypeptide is attached to a solidsupport.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound BLyS binding polypeptide accordingto the present invention. After BLyS binds to a specific BLyS bindingpolypeptide, the unbound serum components are removed by washing,reporter-labeled anti-BLyS binding polypeptide antibody is added,unbound anti-BLyS binding polypeptide antibody is removed by washing,and a reagent is reacted with reporter-labeled anti-BLyS bindingpolypeptide antibody to bind reporter to the reagent in proportion tothe amount of bound BLyS binding polypeptide on the solid support.Typically, the reporter is an enzyme which is detected by incubating thesolid phase in the presence of a suitable fluorometric, luminescent orcalorimetric substrate.

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated BLyS binding polypeptides.

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant BLyS, and a reporter-labeled anti-BLyS bindingpolypeptide antibody for detecting surface-bound anti-BLyS bindingpolypeptide.

Methods of Screening for BLyS Binding Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind BLyS, and the BLyS bindingmolecules identified thereby. This method comprises the steps of:

-   (a) contacting BLyS or BLyS-like polypeptide with a plurality of    molecules; and-   (b) identifying molecule(s) that binds the BLyS or BLyS-like    polypeptide.

The step of contacting the BLyS protein or BLyS-like protein with theplurality of molecules may be effected in a number of ways. For example,one may contemplate immobilizing BLyS target on a solid support andbringing a solution of the plurality of molecules in contact with theimmobilized BLyS target. Such a procedure would be akin to an affinitychromatographic process, with the affinity matrix being comprised of theimmobilized BLyS protein or BLyS-like polypeptide. The molecules havinga selective affinity for the BLyS or BLyS-like polypeptide can then bepurified by affinity selection. The nature of the solid support, processfor attachment of the BLyS or BLyS-like polypeptide to the solidsupport, solvent, and conditions of the affinity isolation or selectionare largely conventional and well known to those of ordinary skill inthe art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” using a BLyS target protein, optionally in the presence ofan inducer should one be required for expression, to determine if anyselective affinity interaction takes place between the BLyS targetprotein and the individual clone. Prior to contacting the BLyS targetprotein with each fraction comprising individual polypeptides, thepolypeptides could first be transferred to a solid support foradditional convenience. Such a solid support may simply be a piece offilter membrane, such as one made of nitrocellulose or nylon. In thismanner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for BLyS orBLyS-like polypeptide. Furthermore, the amino acid sequence of thepolypeptide having a selective affinity for the BLyS protein orBLyS-like protein can be determined directly by conventional means, orthe coding sequence of the DNA encoding the polypeptide can frequentlybe determined more conveniently. The primary amino acid sequence canthen be deduced from the corresponding DNA sequence. If the amino acidsequence is to be determined from the polypeptide itself, one may usemicrosequencing techniques. The sequencing technique may include massspectroscopy.

In certain situations, it may be desirable to wash away any BLyS orBLyS-like polypeptide, or alterntatively, unbound polypeptides, from amixture of BLyS or BLyS-like polypeptide and the plurality ofpolypeptides prior to attempting to determine or to detect the presenceof a selective affinity interaction. One or more such a wash steps maybe particularly desirable when the BLyS or BLyS-like polypeptide or theplurality of polypeptides is bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or non-peptide libraries which can be screened for moleculesthat specifically bind to BLyS. Peptide libraries may be designed suchthat the polypeptides encoded by the libraries are automatically fusedto a polypeptide linker moiety, for example. Many libraries are known inthe art that can be used, e.g., chemically synthesized libraries,recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., Science, 251:767-773 (1991);Houghten et al., Nature, 354:84-86 (1991); Lam et al., Nature, 354:82-84(1991); Medynski, Bio/Technology, 12:709-710 (1994); Gallop et al., J.Medicinal Chemistry, 37(9):1233-1251 (1994); Ohlmeyer et al., Proc.Natl. Acad. Sci. USA, 90:10922-10926 (1993); Erb et al., Proc. Natl.Acad. Sci. USA, 91:11422-11426 (1994); Houghten et al., Biotechniques,13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA,91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA,90:11708-11712 (1993); PCT publication WO 93/20242; and Brenner andLerner, Proc. Natl. Acad. Sci. USA, 89:5381-5383 (1992).

Examples of phage display libraries are described in Scott and Smith,Science, 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990);Christian et al., J. Mol. Biol., 227:711-718 (1992); Lenstra, J.Immunol. Meth., 152:149-157 (1992); Kay et al., Gene, 128:59-65 (1993);and PCT publication WO 94/18318.

In vitro translation-based libraries include but are not limited tothose described in PCT publication WO 91/05058 and Mattheakis et al.,Proc. Natl. Acad. Sci. USA, 91:9022-9026 (1994).

By way of examples of non-peptide libraries, a benzodiazepine library(see, e.g., Bunin et al., Proc. Natl. Acad. Sci. USA, 91:4708-4712(1994) can be adapted for use. Peptoid libraries (Simon et al., Proc.Natl. Acad. Sci. USA, 89:9367-9371 (1992)) can also be used. Anotherexample of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal. (Proc. Natl. Acad. Sci. USA, 91:11138-11142 (1994)).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, Bio/Technology,13:351-360 (1995) list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, Adv. Exp.Med. Biol., 251:215-218 (1989); Scott and Smith, Science, 249:386-390(1990); Fowlkes et al., BioTechniques, 13:422-427 (1992); Oldenburg etal., Proc. Natl. Acad. Sci. USA, 89:5393-5397 (1992); Yu et al., Cell,76:933-945 (1994); Staudt et al., Science, 241:577-580 (1988); Bock etal., Nature, 355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci.USA, 89:6988-6992 (1992); Ellington et al., Nature, 355:850-852 (1992);U.S. Pat. No. 5,096,815; U.S. Pat. No. 5,223,409; and U.S. Pat. No.5,198,346, all to Ladner et al.; Rebar and Pabo, Science, 263:671-673(1993); and PCT publication WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindsBLyS can be carried out by contacting the library members with BLyS orBLyS-like polypeptide immobilized on a solid phase and harvesting thoselibrary members that bind to the BLyS or BLyS-like polypeptide. Examplesof such screening methods, termed “panning” techniques are described byway of example in Parmley and Smith, Gene, 73:305-318 (1998); Fowlkes etal., BioTechniques, 13:422-427 (1992); PCT publication WO 94/18318; andin references cited therein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, Nature, 340:245-246 (1989); Chien etal., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) can be used toidentify molecules that specifically bind to BLyS or BLyS-likepolypeptides.

Where the BLyS binding molecule is a polypeptide, the polypeptide can beconveniently selected from any peptide library, including random peptidelibraries, combinatorial peptide libraries, or biased peptide libraries.The term “biased” is used herein to mean that the method of generatingthe library is manipulated so as to restrict one or more parameters thatgovern the diversity of the resulting collection of molecules, in thiscase peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid, that certain aminoacid positions in a peptide remain fixed (e.g., as cysteine), or thatpositions 4, 8, and 9, for example, of a decapeptide library be limitedto permit several but less than all of the twenty naturally-occurringamino acids. Clearly, many types of biases can be contemplated, and thepresent invention is not restricted to any particular bias. Furthermore,the present invention contemplates specific types of peptide libraries,such as phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a BLyS binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a BLyS binding polypeptide has in the range of 15-100 aminoacids, or 20-50 amino acids.

The selected BLyS binding polypeptide can be obtained by chemicalsynthesis or recombinant expression.

The specific BLyS binding polypeptides disclosed herein were isolatedusing phage display technology, to identify BLyS binding polypeptidesexhibiting particular preselected binding properties. These BLyS bindingpolypeptides were isolated initially by screening nine phage displaylibraries, that is, populations of recombinant bacteriophage transformedto express an exogenous recombinant polypeptide on their surface. Inorder to isolate new polypeptide binding moieties for a particulartarget, such as BLyS, screening of peptide libraries, for example usingphage display techniques, is especially advantageous, in that very largenumbers (e.g., 5×10⁹) of potential binders can be tested and successfulbinders isolated in a short period of time.

In order to prepare a phage library of potential binding polypeptides toscreen for members of the library that are BLyS binding polypeptides, acandidate binding domain is selected to serve as a structural templatefor the polypeptides to be displayed in the library. The phage libraryis made up of polypeptide analogues of this template or “parentalbinding domain.” The parental binding domain is a polypeptide moleculethat may be a naturally occurring or synthetic protein or polypeptide,or polypeptide region or domain of a protein. The parental bindingdomain may be selected based on knowledge of a known interaction betweenthe parental binding domain and a target protein, but this is notcritical. In fact, it is not essential that the parental binding domainhave any affinity for a target at all because its purpose is to providea structure from which a multiplicity of polypeptide analogues (a“library”) can be generated, which multiplicity of polypeptide analogueswill include one or more binding polypeptides that exhibit the desiredbinding and release properties with respect to BLyS target proteins (andany other properties selected).

Knowledge of the exact polypeptide that will serve as the parentalbinding domain, or knowledge of a class of proteins or domains to whichthe parental binding domain belongs can be useful in determining theconditions under which BLyS binding polypeptides optimally bind BLyStarget proteins as well as the conditions under which BLyS bindingpolypeptides optimally release BLyS target proteins. Similarly, thebinding and/or release conditions may be selected with regard to knowninteractions between a binding domain and the BLyS target protein, forexample, to favor the interaction under the binding and/or releaseconditions, or they may be selected without regard to such knowninteractions. Likewise, the parental binding domain can be selectedtaking into account a desired binding and/or release condition or not.It is understood that if the binding domain analogues of a library areunstable under a proposed or desired binding or release condition, nouseful binding polypeptides may be obtained.

In selecting the parental binding domain, the most importantconsideration is how the analogue domains will be presented to the BLyStarget protein, that is, in what conformations the BLyS target and thepolypeptide analogues will contact one another. In preferredembodiments, for example, the polypeptide analogues will be generated byinsertion of synthetic DNA encoding the polypeptide analogue into areplicable genetic package, resulting in display of the domain on thesurface of a microorganism, such as M13 phage, using techniques asdescribed in Kay et al., Phage Display of Peptides and Proteins: ALaboratory Manual (Academic Press, Inc.; San Diego 1996) and U.S. Pat.No. 5,223,409 (Ladner et al.), incorporated herein by reference. Forformation of phage display libraries, it is preferred to use structuredpolypeptides as the parental binding domain or template, as opposed tounstructured, linear peptides. Mutation of surface residues in a proteindomain or polypeptide molecule will usually have little effect on theoverall structure or general properties (such as size, stability, andtemperature of denaturation) of the protein; while at the same timemutation of surface residues may profoundly affect the bindingproperties of the molecule. The more tightly a polypeptide segment isconstrained, the less likely it is to bind to any particular target. Ifit does bind, however, the binding is likely to be tighter and morespecific. Thus, it is preferred to select a parental binding domainwherein the parental polypetide has structure and, thereby in turn,select a structure for the polypeptide analogues of the library, whichis constrained within a framework having some degree of rigidity.

Preferably the protein domain that is used as the template or parentaldomain for generating the library of domain analogues will be a peptidemolecule that is a relatively small protein or polypeptide. Smallpolypeptides offer several advantages over large proteins: First, themass per binding site is reduced. Highly stable protein domains havinglow molecular weights, for example, Kunitz domains (˜7 kilodaltons,kDa), Kazal domains (˜7 kDa), Cucurbida maxima trypsin inhibitor (CMTI)domains (˜3.5 kDa), and endothelin (˜2 kDa), can show much higherbinding per gram than do antibodies (150 kDa) or single chain scFvantibodies (30 kDa). Second, the possibility of non-specific binding isreduced because there is less molecular surface available fornonspecific binding. Third, small polypeptides can be engineered to haveunique tethering sites in a way that is impracticable for largerproteins or antibodies. For example, small proteins and polypeptides canbe engineered to have lysines only at sites suitable for tethering to achromatography matrix. This is not feasible for antibodies. Fourth, aconstrained polypeptide structure is more likely to retain itsfunctionality when transferred (with the structural domain intact) fromone framework to another. For instance, the binding domain structure islikely to be transferable from the framework used for presentation in alibrary, such as displayed on a phage, to an isolated protein removedfrom the presentation framework or immobilized on a chromatographicsubstrate.

In specific embodiments, the BLyS binding polypeptides are immobilized.BLyS binding polypeptide molecules according to the invention may beimmobilized, for example, on chromatographic support materials to formefficient BLyS separation or affinity chromatographic media. ImmobilizedBLyS binding polypeptides have uses that include, but are not limted to,detecting, isolating or removing BLyS target proteins from solutions.One strategy for generating BLyS binding polypeptide molecules that canbe immobilized, for example, on matrices, resins, or supports, involvesselecting appropriate binding domain templates such that BLyS bindingpolypeptide molecules are generated that have one or more amino acidsthat may be used to covalently link the BLyS binding polypeptide to achromatographic resin or substrate to form an affinity resin. Similarly,the N-terminal amino group or the C-terminal carboxyl group of a peptidemolecule may be modified by adding a capping group to render it inert ora functional group, which permits linkage to a support medium. Forexample, the C-terminal carboxyl group of a protein domain may beconverted to an amide or a hydrazide (—NH—NH₂) group for reaction withan aldehyde-functional substrate or other amine-reactive substrate. Thistechnique is preferred. Another preferred modification of BLyS bindingpolypeptides useful for linking a BLyS binding polypeptide molecule to achromatography material is a polypeptide linker comprising, oralternatively consisting of, the amino acid sequencePro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:13).

In one non-limiting example of a screening procedure to obtain BLySbinding polypeptides encompassed by the invention, the phage in a phagedisplay library are contacted with and allowed to bind a BLyS targetprotein that is immobilized on a solid support. Those phage that displaynon-binding polypeptides are separated from those that bind the BLyStarget protein. Any of various techniques known in the art may beapplied to dissociate the bound phage from the immobilized BLyS protein,and to collect and/or amplify the phage and/or their nucleic acidcontents. Using these techniques it is possible to identify a BLySbinding phage that is about 1 in 20 million in the population.Libraries, displaying 10-20 million or more potential binding peptidemolecules each, are rapidly screened to find high-affinity BLyS bindingpolypeptides.

In each round of screening, the diversity of a population falls untilonly efficient binding polypeptides remain, that is, the processconverges. Typically, a phage display library will contain severalclosely related binding polypeptides (10 to 50 different bindingpolypeptides out of 10 million). Indications of convergence includeincreased binding (measured by phage titers) and recovery of closelyrelated sequences. After a first set of binding polypeptide molecules isidentified, the sequence information can be used to design otherlibraries biased for members having additional desired properties, forexample, discrimination between different forms of BLyS (e.g., themembrane form and the soluble form of BLyS) and fragments thereof, ordiscrimination between BLyS and closely related impurities in a feedstream.

Such techniques make it possible not only to screen a large number ofpotential binding polypeptides, but make it practical to repeat thebinding and elution cycles and to build secondary, biased libraries forscreening polypeptide analogue-displaying phage that meet specificcriteria. Using these techniques, a polypeptide analogue biased librarymay be screened to reveal members that bind tightly, that is, have highaffinity for BLyS target protein, under the screening conditions.

In the present invention target BLyS protein molecules were biotinylatedand then bound to streptavidin-coated magnetic particles. Nine phagedisplay libraries of different design were screened for the ability tobind the immobilized BLyS. Each library was characterized by M13 phagedisplaying variegated peptides of different lengths and overallstructure: A library designated TN6/6 (2×10⁸ variants) displayed avariegated 12-mer with two internal invariant cyteines to form a hexamerloop structure. A library designated TN7/4 (2.3×10⁹ variants) presenteda variegated 13-mer having two internal invariant cyteines to form aheptamer loop structure. A library designated TN8/9 (5×10⁹ variants)displayed a variegated 14-mer with two internal invariant cyteines toform an octamer loop structure. A library designated TN9/4 (3.2×10⁹variants) presented a variegated 16-mer having two internal invariantcyteines to form a nonamer loop structure. A library designated TN10/9(2.5×10⁹ variants) displayed a variegated 16-mer with two internalinvariant cyteines to form a decamer loop structure. A librarydesignated TN12/1 (1.4×10⁹ variants) presented a variegated 18-merhaving two internal invariant cyteines to form a dodecamer loopstructure. A library designated as Substrate Phage Library #2, having adiversity of about 2×10⁸ amino acid sequences, was designed to include alinear peptide-variegated region in the display polypeptide consistingof 13 consecutive amino acids, and the display polypeptide designallowed any amino acid residue except cysteine to occur at eachposition. Finally, two commercially available linear phage displaylibraries were also screened, designated PhD 7 and PhD 12, respectively(New England Biolabs). The PhD 7 library displayed a linearrandom-sequence 7-mer; the PhD 12 libary displayed a random-sequence12-mer.

BLyS binding phage were isolated and collected from all of the librariesexcept PhD 7.

After analysis of the sequences isolated from the library screenings,several families of BLyS binding peptides were defined (see, consensussequences A-G and H-L, above). The amino acid sequences of theBLyS-binding “hits” from the first rounds of screening are set forth inTables 1-8 (infra).

In order to obtain BLyS binding polypeptides having an even higheraffinity for BLyS targets, a specialized library was prepared, i.e., aBLyS affinity maturation library, based on variegation of high affinityexamplars of the PhD 12 library (see Example 6). This library wasdesigned to provide a population enriched with polypeptides likely toshow high affinity for BLyS. The selections from this library wereperformed to eliminate, by prolonged competition with soluble eluants ofsoluble BLyS or other BLyS binding polypeptides, all but thepolypeptides having the highest affinity for BLyS. A large family ofhigh affinity BLyS binding polypeptides was isolated from four rounds ofscreening the affinity maturation library, and their amino acidsequences appear in Table 13 (infra).

As it within the scope of the present invention to screen phagelibraries that bind one or more of the various forms of BLyS, thefollowing outlines some assays that may be used in screening for BLySbinding polypeptides that bind the soluble form of BLyS, themembrane-bound form of BLyS, or both the soluble and the membrane-boundforms of BLyS. Assays to determine the specificity of bindingpolypeptides for different forms of a protein are commonly known in theart and may be readily adapted for determining the specificity of BLySbinding polypeptides for different forms of BLyS.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) may be screened in a variety of assays to identifythose BLyS binding polypeptides that specifically bind to the solubleform of BLyS. BLyS binding polypeptides may be assayed in neutralizationassays described herein (see Examples 7 and 8) or otherwise known in theart. For example, BLyS binding polypeptides may be tested for theirability to inhibit soluble BLyS from binding a BLyS receptor. The BLySreceptor used in these assays may be an isolated BLyS receptor (e.g.,BLyS receptor conjugated to agaorose beads) or may be present on thecell surface of cell lines that express BLyS receptors which include,but are not limited to, peripheral CD20+ B cells, IM-9, REH, ARH-77,Namalwa, and RPMI-8226 B cell tumor lines.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants thereof) may be screened in a variety of assays commonly knownin the art to identify those BLyS binding polypeptides that specificallybind to the membrane-bound form of BLyS. For example, BLyS bindingpolypeptides may be assayed for binding BLyS protein present on cellmembranes of cells that express BLyS. Cell lines that express BLyS thatmight be useful for testing BLyS binding polypeptide binding tomembrane-bound form of BLyS include, K-562, HL-60, THP-1, and U937cells.

Aditionally, BLyS binding polypeptides may be screened against cellsengineered to express an “uncleavable” form of BLyS in order todetermine their specificity for the membrane-bound form of BLyS.Mutations in BLyS which may achieve this result include, but are notlimited to, the mutation or deletion of amino acid residues Lys-132and/or Arg-133 of the BLyS sequence shown in SEQ ID NO:173. A typicalmutagenesis might include mutation of one or both of residues Lys-132 orArg-133 to alanine residues. Cells expressing such an “uncleavable” formof BLyS provide a profound reagent to use in assaying the ability ofBLyS binding polypeptides to bind the membrane-bound form of BLyS.

BLyS binding polypeptides (including molecules comprising, oralternatively consisting of, BLyS binding polypeptide fragments orvariants) may be screened in a variety of assays to identify those BLySbinding polypeptides or BLyS binding polypeptide fragments or variantsthat specifically bind to the soluble form and membrane-bound form ofBLyS. This can readily be determined by performing assays to distinguishbinding to the soluble form and assays to distinguish binding to themembrane-bound form (such as the assays described herein or otherwiseknown in the art), and identifying the BLyS binding polypeptides thatbind both forms.

Additionally, BLyS binding polypeptides may be screened for the abilityto inhibit, stimulate or not significantly alter BLyS activity, e.g.,the ability of BLyS: to bind to its receptor (e.g., TACI and BCMA), tostimulate B cell proliferation, to stimulate immunoglobulin secretion byB cells, to activate B cells, to increase B cell lifespan and/or tostimulate a BLyS receptor signaling cascade (e.g., to activatecalcium-modulator and cyclophilin ligand (“CAML”), calcineurin, nuclearfactor of activated T cells transcription factor (“NF-AT”), nuclearfactor-kappa B (“NF-kappa B”; NF-κB), activator protein-1 (AP-1), SRF,extracellular-signal regulated kinase 1 (ERK-1), polo like kinases(PLK), ELF-1, high mobility group I (HMG-I), and/or high mobility groupY (HMG-Y)). Assays that may be used to screen for the effects on BLySactivity are described herein (see, for example, Examples 7, 8, and 12)and are commonly known in the art.

Anti-BLyS Binding Polypeptide Antibodies

Further polypeptides useful herein relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a BLyS bindingpolypeptide (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding). Antibodies include, but arenot limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-id) antibodies (including, e.g., anti-id antibodiesto antibodies), and epitope-binding fragments of any of the above. Theterm “antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass of immunoglobulin molecule. Immunoglobulinsmay have both a heavy and light chain. In specific embodiments, theimmunoglobulin molecules are IgG1. In other specific embodiments, theimmunoglobulin molecules are IgG4. An array of IgG, IgE, IgM, IgD, IgA,and IgY heavy chains may be paired with a light chain of the kappa orlambda forms.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesmay be from any animal origin including birds and mammals. Preferably,the antibodies are human, murine (e.g., mouse and rat), donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 toKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715, WO 92/08802, WO 91/00360, WO 92/05793; Tutt et al., J.Immunol., 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.,148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a BLyS binding polypeptide ofthe present invention which they recognize or specifically bind.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind BLyS binding polypeptides ofthe present invention, and allows for the exclusion of the same.

In further preferred, nonexclusive embodiments, the antibodies (e.g.,anti-idiotypic antibodies) inhibit one or more biological activities ofBLyS through specific binding to BLyS. In more preferred embodiments,the antibody inhibits BLyS-mediated B cell proliferation.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother BLyS binding polypeptide are included. Antibodies that bindpolypeptides with at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 65%, at least 60%, at least55%, and at least 50% identity (as calculated using methods known in theart and described herein) to a BLyS binding polypeptide of the presentinvention are also included in the present invention. Antibodies that donot bind polypeptides with less than 95%, less than 90%, less than 85%,less than 80%, less than 75%, less than 70%, less than 65%, less than60%, less than 55%, and less than 50% identity (as calculated usingmethods known in the art and described herein) to a BLyS bindingpolypeptide of the present invention are also included in the presentinvention. Further included in the present invention are antibodieswhich bind polypeptides encoded by polynucleotides, the complement ofwhich hybridize to a polynucleotides of the present invention understringent hybridization conditions (as described herein). Antibodies ofthe present invention may also be described or specified in terms oftheir binding affinity to a BLyS binding polypeptide. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10 ⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to a BLyS binding polypeptide as determined byany method known in the art for determining competitive binding. Inpreferred embodiments, the antibody competitively inhibits binding tothe BLyS binding polypeptide by at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least50%.

Antibodies of the present invention (e.g., anti-idiotypic antibodies)may act as agonists or antagonists of BLyS or alternatively may notsignificantly alter BLyS mediated activity. For example, the presentinvention includes antibodies (e.g., anti-idiotypic antibodies) whichdisrupt BLyS/BLyS receptor (e.g., TACI and BCMA) interactions eitherpartially or fully. In another example, antibodies of the inventionenhance BLyS/BLyS receptor interactions either partially or fully. Suchactivity may be the result of, for example, the antibody binding to aBLyS binding polypeptide, or alternatively as a result of direct bindingof the antibody (e.g., an anti-idiotypic antibody to BLyS).

Preferrably, antibodies of the present invention bind a BLyS bindingpolypeptide disclosed herein, a portion thereof, or an antibody thatbinds a BLyS binding polypeptide disclosed herein, or a portion thereof.The invention features both BLyS binding polypeptide-specific antibodiesand antibodies that are specific to BLyS binding polypeptide/BLyScomplexes. The invention features antibodies that enhance BLyS/BLySbinding polypeptide binding and/or BLyS/BLyS receptor binding. Theinvention also features antibodies that do not inhibit or reduceBLyS/BLyS binding polypeptide binding and/or BLyS/BLyS receptor binding.The invention also features BLyS binding polypeptide specific antibodiesthat inhibit binding of the BLyS binding polypeptide to BLyS or BLySbinding to BLyS receptor. In specific embodiments, antibodies areprovided that inhibit BLyS activity or BLyS receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody. Receptor activation (i.e., signaling) may be determined bytechniques described herein or otherwise known in the art. For example,receptor activation can be determined by detecting the phosphorylation(e.g., tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra).

The antibodies of the present invention may be used, for purposesincluding, but not limited to, purify, detect, and target the BLySbinding polypeptides of the present invention, including both in vitroand in vivo diagnostic and therapeutic methods. For example, theantibodies have use in immunoassays for qualitatively and quantitativelymeasuring levels of BLyS in biological samples. See, e.g., Harlow etal., Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor 1988).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugated) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396 387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for theantigen. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum. Suchadjuvants are also well known in the art.

According to certain embodiments of the invention, multivalent BLySbinding polypeptides are administered to the host animal. MultivalentBLyS binding polypeptide complexes may be prepared using techniques andmaterials known in the art such as, for example, by cross-linking thepolypeptide to a carrier protein (e.g., bovine serum albumin (BSA),human albumin, keyhole limpet hemocyanin (KLH), or succinylated KLH) byuse of conventional cross-linking reagents.

In specific embodiments multivalent BLyS binding polypeptides areadministered in the form of multiple antigen peptides (MAP) (Tam, J.Imm. Meth., 124:53-61 (1989); Tam, Proc. Natl. Acad. Sci. USA,85:5409-5413 (1988)). In this form, the multivalent BLyS bindingpolypeptide is synthesized on a branching lysyl matrix using solid-phasepeptide synthesis methods. Recognition units in the form of MAP may beprepared by methods known in the art (Tam, 1989, supra; Tam, 1988,supra), or, for example, by a stepwise solid-phase procedure on MAPresins (Applied Biosystems), utilizing methodology established by themanufacturer. MAP peptides may be synthesized comprising (BLyS bindingpolypeptide)₂ Lys₁, (BLyS binding polypeptide)₄ Lys₃, (BLyS bindingpolypeptide)₈ Lys₆ or more levels of branching.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor 1988); Hammerling et al., in Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y. 1981), pp. 563-681 (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

A “monoclonal antibody” may comprise, or alternatively consist of, twoproteins, i.e., a heavy and a light chain.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 9). In a non-limitingexample, mice can be immunized with a polypeptide or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the American TypeCulture Collection (ATCC), to form hybridoma cells. Hybridomas areselected and cloned by limited dilution. The hybridoma clones are thenassayed by methods known in the art for cells that secrete antibodiescapable of binding a polypeptide. Ascites fluid, which generallycontains high levels of antibodies, can be generated by immunizing micewith positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen according tothe invention with myeloma cells and then screening the hybridomasresulting from the fusion for hybridoma clones that secrete an antibodyable to bind a BLyS binding polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH 1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles that carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods, 182:41-50(1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic etal., Gene, 187 9-18 (1997); Burton et al., Advances in Immunology,57:191-280 (1994); PCT international application No. PCT/GB91/01134; PCTpublications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869(1992); and Sawai et al., AJRI, 34:26-34 (1995); and Better et al.,Science, 240:1041-1043 (1988) (said references incorporated herein byreference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shuet al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra etal., Science, 240:1038-1040 (1988). For some uses, including in vivo useof antibodies in humans and in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science, 229:1202(1985); Oi et al., BioTechniques, 4:214 (1986); Gillies et al., J.Immunol. Methods, 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816397, which are incorporated herein by reference intheir entirety. A humanized antibody is an antibody molecule made usingone or more complementarity determining regions (CDRs) from a non-humanspecies antibody that binds the desired antigen and framework regionsfrom a human immunoglobulin molecule. Often, framework residues in thehuman framework regions will be substituted with the correspondingresidue from the CDR donor antibody to alter, preferably improve,antigen binding. These framework substitutions are identified by methodswell known in the art, e.g., by modeling of the interactions of the CDRand framework residues to identify framework residues important forantigen binding and sequence comparison to identify unusual frameworkresidues at particular positions. (See, e.g., Queen et al., U.S. Pat.No. 5,585,089; Riechmann et al., Nature, 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239 400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592 106; EP 519 596; Padlan, Molecular Immunology, 28(4/5):489-498(1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994);Roguskau. et al., Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)), andchain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a binding polypeptide. Monoclonal antibodies directed againstthe antigen can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.,13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent 598 877; U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, each of whichis incorporated by reference herein in its entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (See, Jespers et al., Bio/technology,12:899-903 (1988).)

Further, antibodies to the BLyS binding polypeptides can, in turn, beutilized to generate anti-idiotype antibodies that “mimic” BLyS bindingpolypeptides, using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, FASEB J., 7(5):437-444 (1989) andNissinoff, J. Immunol., 147(8):2429-2438 (1991).) For example,antibodies which bind to and competitively inhibit the binding of BLySbinding polypeptide to BLyS can be used to generate anti-idiotypes that“mimic” the BLyS/BLyS binding polypeptide binding domain and, as aconsequence, bind to and neutralize or enhance BLyS binding to BLySreceptor (e.g., TACI and BCMA). Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens tobind BLyS and/or neutralize or enhance BLyS mediated acitivity. In aspecific embodiment, anti-idiotypic antibodies can be used to bind BLyS,and thereby block its biological activity. In another specificembodiment, anti-idiotypic antibodies can be used to bind BLyS, andthereby enhance its biological activity (e.g., via multimerization ofBLyS).

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent hybridization conditions, e.g., as defined supra, topolynucleotides that encode an antibody, preferably, that specificallybinds to BLyS or a BLyS binding polypeptide.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques,17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method known in theart.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed. (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. 1990) and CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,NY 1993), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol., 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds BLyS or a BLyS binding polypeptide.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:851-855(1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al.,Nature, 314:452-454 (1985)) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.As described supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science, 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Wardet al., Nature, 334:544-54 (1989)) can be adapted to produce singlechain antibodies. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science, 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, (e.g., a heavy or light chain of an antibody or a single chainantibody), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody or portionthereof (preferably containing the heavy or light chain variable domain)has been obtained, the vector for the production of the antibodymolecule may be produced by recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing a protein byexpressing a polynucleotide containing an antibody-encoding nucleotidesequence are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule, or aheavy or light chain thereof, or a heavy or light chain variable domain,operably linked to a promoter. Such vectors may include the nucleotidesequence encoding the constant region of the antibody molecule (see,e.g., PCT publication WO 86/05807; PCT publication WO 89/01036; and U.S.Pat. No. 5,122,464) and the variable domain of the antibody may becloned into such a vector for expression of the entire heavy or lightchain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody. Thus, the invention includes hostcells containing a polynucleotide encoding an antibody, or a heavy orlight chain thereof, or a single chain antibody, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules. Such host-expression systems represent vehiclesby which the coding sequences of interest may be produced andsubsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing antibody coding sequences;yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingantibody coding sequences; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as Escherichia coli, and more preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule, are used for the expression of a recombinant antibodymolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene, 45:101 (1986);Cockett et al., Bio/Technology, 8:2 (1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.,24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. See, e.g., Logan &Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, Bittner et al., Methodsin Enzymol., 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,NSO, 293, 3T3, WI38, and in particular, breast cancer cell lines suchas, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammarygland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418; Wuand Wu, Biotherapy, 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573-596 (1993); Mulligan, Science, 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem., 62:191-217 (1993); May, 1993,TIB TECH 11(5):155-215); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Current Protocols in Molecular Biology, Ausubel et al.,eds. (John Wiley & Sons, NY 1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual (Stockton Press, NY 1990); and CurrentProtocols in Human Genetics, Dracopoli et al., eds. (John Wiley & Sons,NY 1994), Chapters 12 and 13; Colberre-Garapin et al., J. Mol. Biol.,150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol., 3:257(1983)).

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical selectable markers which enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector maybe used which encodes, and is capable of expressing, both heavy andlight chain polypeptides. In such situations, the light chain should beplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature, 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.USA, 77:2197 (1980)). The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

Once an antibody molecule has been produced by an animal, chemicallysynthesized, or recombinantly expressed, it may be purified by anymethod known in the art for purification of an immunoglobulin molecule,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins. Inaddition, the antibodies of the present invention or fragments thereofcan be fused to heterologous polypeptide sequences described herein orotherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanBLyS binding polypeptides of the present invention. For example,antibodies may be used to target the polypeptides of the presentinvention to particular cell types, either in vitro or in vivo, byfusing or conjugating the polypeptides of the present invention toantibodies specific for particular cell surface receptors. Antibodiesfused or conjugated to the polypeptides of the present invention mayalso be used in in vitro immunoassays and purification methods usingmethods known in the art. See e.g., Harbor et al., supra, and PCTpublication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett.,39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc. Natl.Acad. Sci. USA, 89:1428-1432 (1992); Fell et al., J. Immunol.,146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307 434; EP 367 166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA, 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a BLyS bindingpolypeptide may be fused or conjugated to the above antibody portions toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. Further, the BLyS bindingpolypeptides may be fused or conjugated to the above antibody portionsto facilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394 827; Traunecker etal., Nature, 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem., 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties (see, EP-A-232 262). Alternatively, deletingthe Fc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition, 8:52-58 (1995); Johanson etal., J. Biol. Chem., 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA, 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates can be used for modifying a given biological response,the therapeutic agent or drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, anapoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, PCT publicationWO 97/33899), AIM II (See, PCT publication WO 97/34911), Fas Ligand(Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, PCTpublication WO 99/23105), CD40 Ligand, a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin or endostatin; or, biologicalresponse modifiers such as, for example, lymphokines, interleukin-1(“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocytemacrophage colony stimulating factor (“GM-CSF”), granulocyte colonystimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al., eds. (Alan R. Liss, Inc. 1985), pp.243-56; Hellstrom et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery (2nd Ed.), Robinson et al., eds. (Marcel Dekker, Inc.1987), pp. 623-53; Thorpe, “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al., eds., pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody in Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., eds. (Academic Press1985), pp. 303-16; and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982).

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the BLyS bindingpolypeptide. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Current Protocols inMolecular Biology, Ausubel et al., eds. (John Wiley & Sons, NY 1993),which is incorporated by reference herein in its entirety). Exemplaryimmunoassays are described briefly below (but are not intended by way oflimitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,NY 1993) at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,NY 1993) at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley& Sons, NY 1993) at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the diseases, disorders, or conditions disclosed herein.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant BLySexpression and/or activity, including, but not limited to, any one ormore of the diseases, disorders, or conditions described herein.

The treatment and/or prevention of diseases, disorders, or conditionsassociated with aberrant expression and/or activity of BLyS or BLySreceptor includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. The antibodiesof the invention may also be used to target and kill cells expressingBLyS on their surface and/or cells having BLyS bound to their surface.This targeting may be the result of binding of the antibody to BLySbinding polypeptides that have been coadministered, or alternatively,the result of direct binding of the antibody to BLyS. Antibodies of theinvention may be provided in pharmaceutically acceptable compositions asknown in the art or as described herein.

Non-limiting examples of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding BLyS bindingpolypeptides of the present invention that have been coadministered inorder to bind or neutralize BLyS, or by direct cytotoxicity of theantibody, e.g., as mediated by complement (CDC) or by effector cells(ADCC). BLyS binding polypeptides and anti-BLyS binding polypeptideantibodies may be administered either locally or systemically. Some ofthese approaches are described in more detail below. Armed with theteachings provided herein, one of ordinary skill in the art will knowhow to use the antibodies of the present invention for diagnostic,monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoictic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents,antibiotics, and immunoglobulin). Generally, administration of productsof a species origin or species reactivity (in the case of antibodies)that is the same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides of the presentinvention, fragments or regions thereof, for both immunoassays directedto and therapy of disorders related to polypeptides, including fragmentsthereof, of the present invention. Such antibodies, fragments, orregions, will preferably have an affinity for polypeptides, includingfragments thereof. Preferred binding affinities include those with adissociation constant or K_(D) less than 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10 ⁻¹⁰M, 10⁻¹⁰ M, 5×10 ⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Demonstration of Therapeutic or Prophylactic Activity of Antibodies

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic and/or Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a BLyS bindingcompound or pharmaceutical composition, preferably an antibody. In apreferred embodiment, the compound is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side effects). The subject is preferably an animal, includingbut not limited to animals such as cows, pigs, horses, chickens, cats,dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the compound,receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.,262:4429-4432 (1987)), construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of introduction include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The compounds orcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compounds or compositions intothe central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions locally to the area in need oftreatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an antibody, care must be takento use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science, 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp.353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987);Buchwald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl. J.Med., 321:574 (1989)). In another embodiment, polymeric materials can beused (see Medical Applications of Controlled Release, Langer and Wise,eds. (CRC Press, Boca Raton, Fla. 1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball,eds. (Wiley, New York 1984); Ranger and Peppas, J. Macromol. Sci. Rev.Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190(1985); During et al., Ann. Neurol., 25:351 (1989); Howard et al., J.Neurosurg., 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, Langer and Wise, eds.(CRC Press, Boca Raton, Fla. 1974), vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound is a nucleic acid encoding aprotein, the nucleic acid can be administered in vivo to promoteexpression of its encoded protein, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co.,1990). Such compositions will contain a therapeutically effective amountof the compound, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds for use in the methods of the invention can be formulatedas neutral or salt forms. Pharmaceutically acceptable salts includethose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound used which will be effective in thetreatment, inhibition and prevention of a disease or disorder associatedwith aberrant expression and/or activity of a polypeptide can bedetermined by standard clinical techniques. In addition, in vitro assaysmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies may bereduced by enhancing uptake and tissue penetration (e.g., into thebrain) of the antibodies by modifications such as, for example,lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a BLyS binding polypeptide of interest can be usedfor diagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity ofBLyS. The invention provides for the detection of aberrant expression ofBLyS, comprising (a) contacting cells or body fluid with a BLyS bindingpolypeptide; (b) assaying the expression of BLyS in cells or body fluidof an individual using one or more antibodies specific to the BLysbinding polypeptide and (c) comparing the level of BLyS expression witha standard BLyS expression level, whereby an increase or decrease in theassayed BLyS expression level compared to the standard expression levelis indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) contacting cells or body fluid with a BLyS bindingpolypeptide; (b) assaying the expression of BLyS in cells or body fluidof an individual using one or more antibodies specific to the BLySbinding polypeptide of interest and (c) comparing the level of BLySexpression with a standard BLyS expression level, whereby an increase ordecrease in the assayed BLyS expression level compared to the standardexpression level is indicative of a particular disorder. With respect tocancer, the presence of a relatively high amount of BLyS in biopsiedtissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

Antibodies can be used to assay BLyS protein levels in a biologicalsample using or routinely modifying classical immunohistological methodsknown to those of skill in the art (e.g., see Jalkanen et al., J. Cell.Biol., 101:976-985 (1985); Jalkanen et al., J. Cell. Biol.,105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I,¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La,¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin.

Techniques known in the art may be applied to label antibodies. Suchtechniques include, but are not limited to, the use of bifunctionalconjugating agents (see, e.g., U.S. Pat. Nos. 5,756,065; 5,714,631;5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139;5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each ofwhich are hereby incorporated by reference in its entirety).

One embodiment of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of BLyS in ananimal, preferably a mammal and most preferably a human. In oneembodiment, diagnosis comprises: (a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to BLyS(e.g., a BLyS binding polyptide) or which specifically binds to amolecule that specifically binds to BLyS (e.g., an anti-BLyS bindingpolypeptide antibody); (b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level);(c) determining background level; and (d) detecting the labeled moleculein the subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system. Asdescribed herein, specific embodiments of the invention are directed tothe use of the antibodies to quantitate or qualitate concentrations ofcells of B cell lineage or cells of monocytic lineage.

It will be understood by those skilled in the art that the size of thesubject and the imaging system used will determine the quantity ofimaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ⁹⁹mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specificpolypeptide. In vivo tumor imaging is described in S. W. Burchiel etal., “Immunopharmacokinetics of Radiolabeled Antibodies and TheirFragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection ofCancer, S. W. Burchiel and B. A. Rhodes, eds. (Masson Publishing Inc.1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In a further embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisorder, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc. andcomparing the results.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include butare not limited to computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Antibody Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody, preferably apurified antibody, in one or more containers. In a specific embodiment,the kits of the present invention contain a substantially isolatedpolypeptide comprising an epitope which is specifically immunoreactivewith an antibody included in the kit. Preferably, the kits of thepresent invention further comprise a control antibody which does notreact with the polypeptide of interest. In another specific embodiment,the kits of the present invention comprise two or more antibodies(monoclonal and/or polyclonal) that recognize the same and/or differentsequences or regions of a polypeptide according to the invention. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide. Thediagnostic kit includes a substantially isolated antibody specificallyimmunoreactive with polypeptide or polynucleotide antigens, and meansfor detecting the binding of the polynucleotide or polypeptide antigento the antibody. In one embodiment, the antibody is attached to a solidsupport. In a specific embodiment, the antibody may be a monoclonalantibody. The detecting means of the kit may include a second, labeledmonoclonal antibody. Alternatively, or in addition, the detecting meansmay include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated protein(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

In another specific embodiment, any of the antibodies listed above areconjugated to a toxin or a label (as described supra). Such conjugatedantibodies are used to kill a particular population of cells or toquantitate a particular population of cells. In a preferred embodiment,such conjugated antibodies are used to kill B cells expressing BLySreceptor on their surface. In another preferred embodiment, suchconjugated antibodies are used to quantitate B cells expressing BLySreceptor on their surface.

In another specific embodiment, any of the antibodies listed above areconjugated to a toxin or a label (as described supra). Such conjugatedantibodies are used to kill a particular population of cells or toquantitate a particular population of cells. In a preferred embodiment,such conjugated antibodies are used to kill monocyte cells expressingthe membrane-bound form of BLyS. In another preferred embodiment, suchconjugated antibodies are used to quantitate monocyte cells expressingthe membrane-bound form of BLyS.

The antibodies of the invention also have uses as therapeutics and/orprophylactics which include, but are not limited to, in activatingmonocytes or blocking monocyte activation and/or killing monocytelineages that express the membrane bound form of BLyS on their cellsurfaces (e.g., to treat, prevent, and/or diagnose myeloid leukemias,monocyte based leukemias and lymphomas, monocytosis, monocytopenia,rheumatoid arthritis, and other diseases or conditions associated withactivated monocytes). In a specific embodiment, the antibodies fixcomplement. In other specific embodiments, as further described herein,the antibodies (or fragments thereof) are associated with heterologouspolypeptides or nucleic acids (e.g. toxins, such as, compounds that bindand activate endogenous cytotoxic effecter systems, and radioisotopes;and cytotoxic prodrugs).

As discussed above, antibodies to the BLyS binding polypeptides can, inturn, be utilized to generate anti-idiotype antibodies that “mimic” theBLyS binding polypeptide, using techniques well known to those skilledin the art. (See, e.g., Greenspan & Bona, FASEB J., 7(5):437-444 (1989),and Nissinoff, J. Immunol., 147(8):2429-2438 (1991)). For example,antibodies which bind to BLyS binding polypeptides and competitivelyinhibit BLyS/BLyS binding polypeptide binding can be used to generateanti-idiotypes that “mimic” the BLyS binding polypeptide/BLyS bindingdomain and, as a consequence, bind to and, for example, neutralize BLyS.Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypescan be used in therapeutic regimens to neutralize BLyS. For example,such anti-idiotypic antibodies can be used to bind BLyS and therebyblock BLyS mediated B cell activation, proliferation, survival and/ordifferentiation.

EXAMPLES

Isolation of BLyS binding polypeptides and their use in accordance withthis invention will be further illustrated below. The specificparameters included in the following examples are intended to illustratethe practice of the invention, and they are not presented to in any waylimit the scope of the invention.

Example 1 Screening of Phage Display Libraries

Streptavidin-coated magnetic beads (Dynal M-280) were chosen forpresentation of the target during screening because of their superiorbinding capacity compared to that of a 96 well plate. The bindingcapacity of the beads for biotinylated antibodies was 5-10 μg/mg ofbeads as stated by the manufacturer. For this study and the screening tofollow, 5 μg of biotinylated recombinant BLyS (obtained from HumanGenome Sciences, Inc.) was allowed for each mg of beads. This amount ofbiotinylated BLyS represents a 10-fold excess of target, for saturationof the beads. Unbound BLyS was washed away. Bound biotinylated BLyS wasconfirmed with detection using Mab 16C9 (murine anti-BLyS, Human GenomeSciences) primary antibody and a goat anti-mouse HRP conjugate as thesecondary antibody. An irrelevant monoclonal antibody (anti-TNFα) wasused to probe a second set of beads to control for nonspecific binding.The color reagent TMB was used and the assay read at OD 630 nm.

Nine phage display libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9,TN12/1, and Substrate Phage #2 (Dyax Corp., Cambridge, Mass. (US)), andPhD7 and PhD12 (New England Biolabs), were screened for BLyS binders.The makeup of these libraries was as follows:

The TN6/6 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:14) andproviding 2.0×10⁸ peptide diversity.

The TN7/4 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:15) andproviding 2.3×10⁹ peptide diversity.

The TN8/9 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:16)and providing about 5×10⁹ peptide diversity.

The TN9/4 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ IDNO:17) and providing about 3.2×10⁹ peptide diversity.

The TN10/9 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ IDNO:18) and providing 2.5×10⁹ peptide diversity.

The TN12/1 phage display library was composed of recombinant M13 phagedisplaying variegated peptides with the potential to form loopstructures based on a polypeptide template having the structureXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa(SEQ ID NO:19) and providing 1.4×10⁹ peptide diversity.

Substrate Phage Library #2 was composed of recombinant M13 phagedisplaying a polypeptide insert of approximately 80 amino acids, havingtwo streptavidin binding domains, a linear variegated segment ofthirteen amino acids where all amino acids except Cys were permitted ateach position, and a Factor Xa cleavage site, linked together withpeptide linkers. This library provided a diversity of 2×10⁸ displaypolypeptides.

Libraries PhD7 and PhD 12 were composed of recombinant M13 phagedisplaying randomized linear seven- and twelve-amino acid peptides,respectively.

Screening was performed as described in PCT/US01/[______], entitled“Binding Polypeptides for B Lymphocyte Stimulator Protein (BLyS)”, filedconcurrently herewith.

At the conclusion of screening individual phage isolates were randomlyselected and tested by ELISA for binding to BLyS. The same isolates weresubmitted for DNA sequence analysis to identify the nucleotide anddeduced amino acid sequence of the displayed peptide. Isolates were alsotested for their ability to bind to recombinant BLyS in feed streams ofCHO supernatant and Sf9 supernatant (supplied by Human Genome Sciences,Inc.).

Each isolate was tested for binding to BLyS by standard ELISA techniqueswhere bound phage were detected with a monoclonal anti-phageantibody/HRP conjugate.

Amino acid sequences of the displayed peptides were derived fromsequencing the phage isolate DNA inserts. Sequence data from the phageisolates were grouped by library and sorted according to the degree ofsimilarity. The BLyS binding phage isolate peptides are shown in Tables1-8 below. These peptides represent the translation of the DNA sequencesacross the varied regions of the genes encoding the phage displayfusion/peptide. TABLE 1 TN6/6 Library BLyS-binding Sequences PhageIsolate Amino Acid Sequence SEQ ID NO: 453-01-B06 HLRCWSTNCRYD 20453-01-A04 VMDCLINRCDTV 21

TABLE 2 TN7/4 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-01-B04 KSKCFFPWECQQA 22 453-01-D11 AMKCYFPWECANG23 453-01-A05 NVACYFPWECHHP 24 453-01-D01 NAPCYFPWECFSI 25 453-01-D03SVNCWFPWECVGN 26 453-01-A08 KEPCYFYWECAVS 27

TABLE 3 TN8/9 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-01-D04 DTNCDLLTKMCGPQ 28 453-01-C06GTPCDLLTKLCLLW 29 453-01-D10 MSECDLLTKICLMG 30 453-01-B07 VPFCDLLTKHCFEA31 453-01-B09 VPFCDLLTKHCFEA 32 453-01-C02 WSACDLLTKQCVQV 33 453-01-A06-DGCDELTKICGMK 34 453-01-B03 KSWCDELTKVCFDP 35 453-01-B11 KWMCDELTKQCQYV36 453-01-A02 MKYCDELTKICVGW 37 453-01-B05 YFQCDELTKMCWQK 38 453-01-A11AMHCDKLTKHCKFH 39 453-01-A03 VPYCDKLTKICQW- 40 453-01-A07 EVFCDVLTKVCFHD41 453-01-C09 KPKCDVLTKMCDWL 42 453-01-B02 TQHCDVLTKQCFTI 43 453-01-C01GHFCDRLTKYCFEP 44 453-01-A09 HIQCDRLTKSCLSV 45 453-0l-D05 IKACDILTKVCWPP46 453-01-A01 QFDCDPLTKYCGEF 47 453-01-C07 KMYCDHLTGYCWPE 48 453-01-C11MQSCDILTGYCFKR 49 453-01-D12 GPWCDILTGFCLAQ 50 453-01-C04 SVRCDLLTGWCPVW51 453-01-B10 PADCDPLTNICFWK 52 453-01-D02 TNVCDPLTNVCFMN 53 453-01-C05EHWCDDLTHLCFRL 54 453-01-D08 GYWCDVLTNNCWKI 55 453-01-C10 LYNCDYLTRLCFEP56 453-01-C08 HVDCLLHPKACYKY 57 453-01-D07 VQDCLLHPKACQMQ 58 453-01-D09KFDCLLKPMFCSNH 59 453-01-C12 FADCLIHPKSCKPL 60 453-01-D06 HGNCYPFPWECESK61 453-01-B01 MIIVLLLLRFAISR 62 453-01-A12 SLLVIFLLIGAGSL 63

TABLE 4 TN9/4 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-01-G06 FHPCDMLTGIWCQPN 64 453-01-H01SKRCDLLTKMWCETE 65 453-01-F02 TKFCDRLTMPKCVWK 66 453-01-E03NTFCPDPLTGRCVNP 67 453-01-E11 DWTCDPLFHRECIFE 68 453-01-H09PQPCDLLFEKKCSIK 69 453-01-H02 RWHCDMLINPSCLPD 70 453-01-E04KIQCDIVNLSSCVYP 71 453-01-G11 LNACDIVHPNYCSGM 72 453-01-F01AKACSIVNLESCEYL 73 453-01-H06 RQACSIITPWGCPIP 74 453-01-F10ADNCTVATLDFCYWT 75 453-01-E05 KPECNITKPQFCFGE 76

TABLE 5 TN10 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-01-H07 -NNCQWDELTSMCDPF 77 453-01-F05SRLCHMDELTHVCVHF 78 453-01-F09 SRPCQIDELTKACFYN 79 453-01-G09DRVCKLDFLTYNCLNH 80 453-01-F04 HSNCIMDLLTNRCFYD 81 453-01-H03PFNCFHDPLTGLCLHS 82 453-01-F03 YDSCTYDRLTKQCYPS 83 453-01-F07FHDCMYDALLGYCLPY 84 453-01-G08 NRSCDPLTRPKSCGL 85 453-01-G04LSNCDWDDLIRQCLHD 86 453-01-E01 FWDCLFHPNSRYCVLS 87 453-01-E10SRDCLLSPAMAWCGLD 88

TABLE 6 TN12/1 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-01-H05 GGNCYTDSLTKLHFCMGD 89 453-01-H04--MCPRDPLTKAKLCNWH 90 453-01-G03 PNQCQDDLTKQWYSCHYH 91 453-01-F11FDMCFDALTKQNFYCRFH 92 453-01-F06 RNMCVDRLTKLQHGCEGA 93 453-01-G07DPECLTSFDRLTKMCWPW 94 453-01-H11 DDECHYDYLTHYMRCDYR 95 453-01-G05FGGCNIDLLTNTMMCHRN 96 453-01-G10 HGPCYWDELTMQWHCNHH 97 453-01-H12GAMCVDLLTYTFRPCMYA 98 453-01-E07 SNKCWDELTHAWAECGRF 99 453-01-E09RPVCYKGYDILTTQCMPW 100 453-01-G01 PSRCWFDLLFNKFVCKRN 101 453-01-H08RSGCVYDMLLMTMYCPSN 102 453-01-H10 SNRCEGDQLMRPPSCRHL 103 453-01-F08YRMCWWDDLLRGFVCDFH 104 453-01-E06 HDGCYDELLYRWTRCEHR 105 453-01-E08WAWCFDELVQRYFTCFDH 106 453-01-E02 LPECRQYFPWEKQVCSYW 107

TABLE 7 PhD 12 Library BLyS-binding Sequences Phage Isolate Amino AcidSequence SEQ ID NO: 453-02-B05 VHYDSLTKMWTR 108 453-02-D09 FTDPLTKMSLHS109 453-02-C12 GYDVLTKLYFVP 110 453-02-A05 YYDRLTKLYSSM 111 453-02-B06L?KDPLTKLYIS 112 453-02-A04 GYDVLTKL?FVP 113 453-02-B03 RLYDPLTKLVLS 114453-02-B01 MFDPLTKIAFPA 115 453-02-D04 FYDSLTKTNLRD 116 453-02-B02GIYDKLTRAWLP 117 453-02-B08 KYDPLTRAR?PL 118 453-02-D06 YIDQLTRLSLPS 119453-02-A09 HqTFDILTRLHF 120 453-02-B04 WQFDVLTRSWTP 121 453-02-A02GAAYDHLTRTWL 122 453-02-D05 YFDQLTHLSIKK 123 453-02-A06 AWDPLTMLVLPW 124453-02-D03 ALWMDPLTGLAF 125 453-02-B12 WQFDVLT?SWTP 126 453-02-A01WTDPLTHMEIYH 127 453-02-C04 WTDSLTGLWFPD 128 453-02-C05 YTDPLTGIV?PF 129453-02-D08 YWDKLTMLHLGV 130 453-02-D02 YYDFLTRTVLPS 131 453-02-A03RLDPLSKNDFPR 132 453-02-A11 LRYDPLLKS?IY 133 453-02-D07 LRYDPLLKSYIY 134453-02-A07 YFDQFTHLSIKK 135 453-02-C08 YFDQ?THLSIKK 136

TABLE 8 Substrate Phage Library BLyS-binding Sequences Phage IsolateAmino Acid Sequence SEQ ID NO: 453-02-E04 EHYYTDPLTGARI 137 453-02-F01EHY?TDPLTGARI 138 453-02-E09 EHYSTDPLTGARI 139 453-02-E07 EHYYTDPL?G?RI140 453-02-G05 EHYYTDPL?G?R? 141 453-02-G09 EHYYTDPL?GAR? 142 453-02-E06EH?YTDPLNGAR? 143 453-02-E05 EHYYNDPLNGAR? 144 453-02-F04 ?H?YNDPLNGAR?145 453-02-G07 KPYYDPITKMTHH 146 453-02-F06 KPYYDPITKMSHH 147 453-02-E08KPYYDPISKMTHH 148 453-02-G08 KP??DPISKMTHH 149 453-02-E01 QIGYDELTKAWVT150 453-02-G02 QLGYDELTKAWVT 151 453-02-H06 KIDEL?MQNIIIW 152 453-02-F08DHTDPLIQGLTKR 153 453-02-H01 WHDPLKHMHFHHE 154 453-02-F03 KHIDMETGLILQN155 453-02-G03 MQVDPETGLKYEH 156 453-02-E03 ?LDQHVN???YQS 157 453-02-F10E???T??LTGAR? 158 453-02-F02 GPYNI?RL?GEr? 159 453-02-E02 HIKMLHQGSFVGV160 453-02-H08 HPTNT??HQ?VYS 161 453-02-H05 HRGQV??LNGMv? 162? = amino acid unknown (all tables)lower case = amino acid identity probable but not completelycharacterized

Example 2 Immobilization of BLyS Binding Polypeptides on Sepharose-4FFBeads

On the basis of the above results, six display phage sequences werechosen for further study:

TN7-01-A08 (SEQ ID NO:27), TN8-01-B07 (SEQ ID NO:31), TN10-01-F05 (SEQID NO:78), TN12-01-H05 (SEQ ID NO:89), PhD-02-C04 (SEQ ID NO:128), andPhD-02-C12 (SEQ ID NO:110). In order to develop a suitable BLyS affinityligand, the identified display peptides were synthesized to order by acommercial vendor, with slight modifications:

Two amino acids of leader were added to each binding peptide at theN-terminus, in order to avoid leaving a free amine at the first aminoacid of the sequence corresponding to the variegated region of the phagedisplay template; the N-terminus was acetylated to preventimmobilization of the peptide to the chromatographic matrix through thatposition; a C-terminal linker was added (i.e., -PGPEGGGK; SEQ ID NO:13);and any internal lysines in the peptide were blocked with the group:ivDde (i.e., 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine). This group was intact on the finished synthesizedpeptides and was removed after immobilization or fluorescein labeling.As an alternative modification, peptides with internal lysines were alsosynthesized with C-terminal hydrazide functional groups, which could beimmobilized onto activated aldehyde chromatographic media.

The peptides were immobilized onto NHS-activated SEPHAROSE-4 Fast Flowagarose media (Pharmaceia) at ligand densities targeted to 2 μmol/ml.Actual ligand densities of peptides on the media ranged from 0.76μmol/ml to 1.98 μmol/ml, as determined by amino acid analysis ofimmobilized peptide. All but one peptide was immobilized in aqueousconditions of 100 mM KH₂PO₄/150 mM NaCl/0.05% Tween 20, pH 7.5. Forsolubility reasons, the peptide DX217 (see, Table 9, below) wasimmobilized in 30% dimethyl formamide(DMF)/100 mM KH₂PO₄/150 mMNaCl/0.05% Tween 20. pH 7.5. Immobilization reactions were allowed toproceed for 2 hours at ambient temperature, followed by brief washingwith pH 7.5 buffer. The Fast Flow SEPHAROSE media was then allowed totumble at ambient temperature overnight to hydrolyze remaining NHSesters after which the media was washed to remove any unbound peptide. Asolution of 2% hydrazine/DMF was used to de-block ligands containingivDde-lysine. Media was then further washed with aqueous buffers andstored at 4° C. until packed into columns. Table 9 shows the sequencesof the synthesized peptides and their measured densities on the agarosemedia. TABLE 9 BLyS Binding Peptides Synthesizes as Affinity LigandsPeptide Isolate Sequence SEQ ID Name source (potential disulfide loopunderlined) NO: DX212 01-A08 Ac-AGKEPCYFYWECAVSGPGPEGGGK 163 DX21401-B07 Ac-AGVPFCDLLTKHCFEAGPGPEGGGK 164 DX216 01-F-5Ac-GSSRLCHMDELTHVCVHFAPPGPEGGGK 165 DX217 01-H05Ac-GDGGNCYTDSLTKLHFCMGDEPGPEGGGK 166 DX219 02-C12Ac-GYDVLTKLYFVPGGPGPEGGGK 167 DX221 02-C04 Ac-WTDSLTGLWFPDGGPGPEGGGK 168Ac denotes N-terminal acetylationBLyS-Ligand Affinity Determination (Overview of Procedure)

Dissociation constants between the synthetic peptides and BLyS (free insolution) were measured by fluorescence anisotropy (FA). In theseexperiments, the concentration of the fluorescein-labeled peptide isheld constant and the BLyS protein concentration was varied. Theobserved change in anisotropy is fit to the following equation vianonlinear regression to obtain the apparent K_(D).${Peptide} + {{{BLyS}\underset{K_{D}}{\longleftrightarrow}{Peptide}} \cdot {BLyS}}$$r_{obs} = {r_{free} + {\left( {r_{bound} - r_{free}} \right)\frac{\left( {K_{D} + {BLYS} + P} \right) - \sqrt{\left( {K_{D} + {BLYS} + P} \right)^{2} - {4 \cdot {BLYS} \cdot P}}}{2 \cdot P}}}$where:r_(obs)=observed anisotrpy, r_(free)=anisotropy of free peptide,r_(bound)=anisotropy of bound peptide, K_(D)=dissociation constant,BLyS=total BLyS concentration, and P=total fluorescein labeled peptideconcentration.

Binding reactions containing 50 nM fluorescein-labeled peptide and avaried concentration of BLyS in a volume between 10 and 20 μL per wellwere performed in 384 well microplates. Reactions were assayed using aTecan Polarion fluorescence polarization plate reader. Cross-competitionstudies between peptides were performed using 50 nM fluorescein-labeledpeptide and 1-2 μM BLyS in the presence and absence of 100 μM unlabeledpeptide. The influence of pH on the observed K_(D) was investigated atpH 6.0 using the primary binding buffer [15 mM sodium citrate, 120 mMNaCl, 0.01% Tween 20] and at pH 9.0 using 200 mM sodium bicarbonate, 125mM sodium chloride. Other buffers in which dissociation constants ofBLyS Binding polypeptides were detremined include: [pH 6.0, 0.01%Tween], [pH 6.0, 0.1% gelatin], [pH5.0, 0.01% Tween], [pH9.0, 0.1%Tween], [pH6.0, 15% ethylene glycol, 0.01% Tween], ], [pH5.0, 15%ethylene glycol, 0.01% Tween], and [pH9.0, 15% ethylene glycol, 0.01%Tween]. All six of the peptides (DX212, DX214, DX216, DX217, DX219, andDX221) bound BLyS in solution with approximately the same affinity(K_(D)=0.5-2 μM). Cross-competition studies demonstrated that allpeptides compete with each other for BLyS binding, which suggests thatthey all bind to the same site on BLyS.

Example 3 Design of modified BLyS Binding Peptides

Once a promising BLyS binding polypeptide has been isolated,improvements to that polypeptide can be made by changing, adding orremoving individual or multiple amino acid residues from thepolypeptide. Amino acid substitutions can be conservative or nonconservative. Conservative amino acids exchanges include, for example,the exchange of aromatic residues (e.g., phenylalanine, tryptophan, andtyrosine) for one another, the exchange of hydrophobic residues (e.g,leucine, isoleucine, and valine) for one another, the exchange of polarresidues (e.g., glutamine and asparagine) for one another, the exchangeof acidic residues (e.g., arginine, lysine, and histidine) for oneanother, and the exchange of small residues (e.g., alanine, serine,threonine, methionine, and glycine) for one another, the exchange ofaromatic residues for one another. Additionally, nonclassical aminoacids, chemical amino acid analogs, or chemically modified classicalamino acids can be introduced as a substitution or addition to a BLySbinding polypeptide of the invention. Non-classical amino acids include,but are not limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid (Dbu), 4-aminobutyric acid (bAbu),2-aminobutyric acid (Abu), 6-amino hexanoic acid (epsilon-Ahx),2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib),3-aminopropanoic acid (bAla), ornithine (Orn), norleucine (Nle),norvaline (Nva), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp),sarcosine (MeGly), citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Ca-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. By way of example, four modified peptides based on the DX212sequence have been designed:

1. Ac-AGK(Ac)EPCYFYWECAVSGPGPEGGGK (SEQ ID NO:169)—internal lysine sidechain acetylated;

2. Ac-AGREPCYFYWECAVSGPGPEGGGK (SEQ ID NO:170)—arginine substitution;

3. Ac-AGQEPCYFYWECAVSGPGPEGGGK (SEQ ID NO:171)—glutamine substitution;

4. Ac-AGNleEPCYFYWECAVSGPGPEGGGK (SEQ ID NO:172)—norleucinesubstitution. Ac denotes N-terminal acetylation.

Example 4 Biacore Analysis of the Affinity of BLyS Binding Polypeptides

Binding of BLyS binding polypeptides to BLyS, for example, can beanalyzed by BIAcore analysis. Either BLyS (or another antigen for whichone wants to know the affinity of a BLyS binding polypeptide) or BLySbinding polpeptide can be covalently immobilized to a BIAcore sensorchip (CM5 chip) via amine groups usingN-ethyl-N′-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimidechemistry. Various dilutions of BLyS binding polypeptides or BLyS (orother antigen for which one wants to know the affinity of a BLyS bindingpolypeptide), respectively are flowed over the derivatized CM5 chip inflow cells at 15 microlters/min. for a total volume of 50 microliters.The amount of bound protein is determined during washing of the flowcell with HBS buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 3.4 mM EDTA,0.005% surfactant P20). Binding specificty for the protein of inerest isdetermined by competition with soluble competitor in the presence theprotein of ineterest.

The flow cell surface can be regenerated by displacing bound protein bywashing with 20 microliters of 10 mM glycine-HCl, pH2.3. For kineticanalysis, the flow cells are tested at different flow rates anddifferent polypetide densities on the CM5 chip. The on-rates andoff-rates can be determined using the kinetic evaluation program inBIAevaluation 3 software.

Example 5 BLyS Binding Polypeptide Neutralization of Murine SplenocyteProliferation

To determine if an BLyS binding polypeptide inhibits BLyS mediated Bcell proliferation, a splenocyte proliferation assay can be performedBriefly, murine splenocytes are isolated by flushing spleen withcomplete medium using a 25 g needle and 10 ml of complete medium (RPMI1640 with 10% FBS containing 100 U/ml penicillin, 100 μg/mlstreptomycin, 4 mM glutamine, 5×10⁻⁵M β-mercaptoethanol). The cells arepassed through a 100 micron nylon filter to remove cell clumps. The cellsuspension is then separated by gradient centrifugation at 400×g for 25minutes at room temperature (one 15 ml conical tube/spleen; 3 ml Ficol,10 ml cell suspension/spleen; Ficol 1083 from Sigma). The recoveredcells are washed 3 times in complete medium and counted. Recovered cellsare then diluted to a concentration of 3×10⁶/ml in complete mediumcontaining a 3× concentration of SAC (3×=1:33,333 dilution of stockStaph. aureus Cowan strain; Calbiochem).

For each BLyS binding polypeptide, 50 microliters of dilutions at 30μg/ml, 3.0 μg/ml, and 0.3 μg/ml concentrations are aliquotted intoindividual wells of a 96 well plate in triplicate. Suitable positivecontrols, such as, for example monoclonal antibody 15C10, can also beused. Medium containing no BLyS binding polpeptide is used as negativecontrol. BLyS protein is diluted in complete medium to concentrations of300 ng/ml, 90 ng/ml and 30 ng/ml. 50 microliters of each of the BLySdilutions were then added to the BLyS binding polypeptide dilutionseries in the plates. The plate containing the BLyS binding polypeptideand BLyS dilutions are then incubated for 30 minutes at 37° C., 5% CO₂,after which 50 microliters of the splenocyte cell suspension containingSAC is added to all wells. The plates are then incubated for 72 hours(37° C., 5% CO₂).

After 72 hours, each well is supplemented with 50 μl of complete mediumcontaining 0.5 μCi of ³H-thymidine (6.7 Ci/mM; Amersham) and cells areincubated for an additional 20-24 hours at (37° C., 5% CO₂). Followingincubation cells are harvested using a Tomtec Cell Harvester and filterscounted in a TopCount Scintillation counter (Packard).

Example 6 In Vitro Screening of BLyS Antagonists

The bioassay for assessing the effects of putative BLyS antagonists isperformed in triplicate in 96 well format by mixing equal volumes ofBLyS, responder cells, and putative antagonist each of which is preparedas a 3× stock reagent.

B-lymphocytes are purified from human tonsil by MACS (anti-CD3depletion), washed, and resuspended in complete medium (CM) (RPMI 1640with 10% FBS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 4mM glutamine, 5×10E-5 M beta-mercaptoethanol) at a concentration of3×10e6 cells/mL. Staphylococcus aureus, Cowan I (SAC, CalBiochem) isadded to cells at 3× concentration (3×=1:33,333 dilution of stock).

Meanwhile, eight serial dilutions (3-fold) of potential antagonists areprepared in CM such that the diluted antagonists are at 3× the finalconcentrations to be tested in the assay. BLyS binding polypeptides areroutinely tested starting at a final concentration of 10 μg/mL and goingdown to about 1.5 ng/mL.

Human rBLyS was prepared in CM to 3× concentration (3×=300 ng/mL, 30ng/mL, and 3 ng/mL) in CM. Potential inhibitors are routinely tested atseveral concentrations of BLyS to avoid false negatives due tounexpectedly low affinity or antagonist concentration.

Fifty microliters of diluted antagonist and 50 μL of diluted BLyS areadded to the putative antagonist dilution series. Cells are thenincubated for 72 hours (37° C., 5% CO₂) in a fully humidified chamber.After 72 hrs., the cells are supplemented with 0.5 μCi/well 3H-thymidine(e.g., 6.7 Ci/mmol) and incubated for an additional 24 hours. Plates areharvested using a Tomtec Cell Harvester and filters counted in aTopCount Scintillation counter (Packard).

Example 7 Protein Fusions of BLys Binding Polypeptides

BLyS binding polypeptides of the invention are optionally fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of BLyS binding polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See, EP A 394 827; Traunecker et al., Nature,331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albuminincreases the half-life time in vivo. Nuclear localization signals fusedto BLyS binding polypeptides can target the protein to a specificsubcellular localization, while covalent heterodimer or homodimers canincrease or decrease the activity of a fusion protein. Fusion proteinscan also create chimeric molecules having more than one function.Finally, fusion proteins can increase solubility and/or stability of thefused protein compared to the non-fused protein. All of the types offusion proteins described above can be made using techniques known inthe art or by using or routinely modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow (SEQ ID NO:447). These primers also preferably contain convenientrestriction enzyme sites that will facilitate cloning into an expressionvector, preferably a mammalian expression vector.

For example, if the pC4 (Accession No. 209646) expression vector isused, the human Fc portion can be ligated into the BamHI cloning site.Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and BLyS binding polynucleotide is ligated into this BamHIsite. Note that the polynucleotide is cloned without a stop codon,otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc Region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCG(SEQ ID NO:449)TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 8 Isolation of scFV Molecules Recognizing BLyS BindingPolypeptides

Naturally occuring V-genes isolated from human PBLs are constructed intoa large library of antibody fragments which contain reactivities againstpolypeptides of the present invention to which the donor may or may nothave been exposed (see, e.g., U.S. Pat. No. 5,885,793, incorporatedherein by reference in its entirety).

Rescue of the Library

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO 92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harbouring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of Δ gene3 helper phage (M13 Δ gene III, see WO 92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 minutes and the pellet resuspended in 2 liters of 2×TYcontaining 100 ug/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phage are prepared as described in WO92/01047.

M13 Δ gene III is prepared as follows: M13 Δ gene III helper phage doesnot encode gene III protein, hence the phage(mid) displaying antibodyfragments have a greater avidity of binding to antigen. Infectious M13 Δgene III particles are made by growing the helper phage in cellsharboring a pUC 19 derivative supplying the wild type gene III proteinduring phage morphogenesis. The culture is incubated for 1 hour at 37°C. without shaking and then for a further hour at 37° C. with shaking.Cells are pelleted (IEC-Centra 8, 4000 revs/min. for 10 min.),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100mg/ml or 10 mg/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 1013 TU of phage are applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used toinfect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 μg/ml ampicillin. The resultingbacterial library is then rescued with Δ gene III helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders

Eluted phage from the 3rd and 4th rounds of selection are used to infectE. coli HB 2151 and soluble scFv is produced (Marks et al., 1991) fromsingle colonies for assay. ELISAs are performed with microtitre platescoated with either 10 pg/ml of the polypeptide of the present inventionin 50 mM bicarbonate, pH 9.6. Clones positive in ELISA are furthercharacterized by PCR fingerprinting (see, e.g., WO 92/01047) and then bysequencing.

Additionaly, scFvs may be converted to complete Ig molecules usingtechniques which are commonly known in the art.

Example 9 Production of an Anti-BLyS Binding Polypeptide Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing BLyS binding polypeptides are administered toan animal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of BLyS bindingpolypeptide is prepared and purified to render it substantially free ofnatural contaminants which is then conjugated to a carrier molecule suchas keyhole limpet hemocyanin (KLH), suucinylated KLH, or chicken gammaglobulin (CGG). Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or BLyS protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology.(Kohler et al., Nature, 256:495 (1975); Kohler et al., Eur. J. Immunol.,6:511 (1976); Kohler et al., Eur. J. Immunol., 6:292 (1976); Hammerlinget al., in Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y.1981), pp. 563-681.) In general, such procedures involve immunizing ananimal (preferably a mouse) with BLyS binding polypeptide or, morepreferably, with a secreted BLyS binding polypeptide-expressing cell.Such cells may be cultured in any suitable tissue culture medium;however, it is preferable to culture cells in Earle's modified Eagle'smedium supplemented with 10% fetal bovine serum (inactivated at about56° C.), and supplemented with about 10 g/l of nonessential amino acids,about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2/0), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology, 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the BLyS bindingpolypeptide.

Alternatively, additional antibodies capable of binding to BLyS bindingpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the BLyS bindingpolypeptide-specific antibody can be blocked by BLyS bindingpolypeptide. Such antibodies comprise anti-idiotypic antibodies to theBLyS binding protein-specific antibody and can be used to immunize ananimal to induce formation of further BLyS binding polypeptide-specificantibodies.

It will be appreciated that Fab and F(ab′)₂ and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). Alternatively, secreted BLySbinding protein-binding fragments can be produced through theapplication of recombinant DNA technology or through syntheticchemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science, 229:1202 (1985); Oi et al., BioTechniques, 4:214 (1986);Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171 496;Morrison et al., EP 173 494; Neuberger et al., WO 86/01533; Robinson etal., WO 87/02671; Boulianne et al., Nature, 312:643 (1984); Neuberger etal., Nature, 314:268 (1985).)

Example 10 BLyS-Induced Signalling in B Cells

Total RNA was prepared from tonsillar B cells unstimulated or stimulatedwith SAC or SAC plus BLyS (100 ng/mL) for 12 hours. Messenger RNA levelsof ERK-1 and PLK was determined by real time quantitaive PCR using ABI7700 Taqman sequence detector. Amplification primers and probes weredesigned to span the region from nucleotides 252-332 of the human PLKsequence and nucleotides 373 to 446 of the human ERK-1 mRNA (GenBankaccession numbers X75932 and X60188, respectively). For quantitation ofRNA, the comparative delta CT method was used (Perkin-Elmer userBulletin #2 and #4, 1997) using an 18S ribosomal RNA probe as endogenousreference. Expression levels were characterized relative to observedlevels in unstimulated B-cells.

Example 11 Affinity Maturation of BLyS Binding Polypeptides

In order to identify high affinity BLyS-binding polypeptides, a BLySAffinity Maturation Library (BAML) was designed around a 14-mer linearpeptide template sequence having fixed amino acid residues at 5 of the14 positions. 3 of the 5 fixed residues corresponded to a highlyconserved DxLT tetrapeptide amino acid motif (SEQ ID NO:446) isolatedfrom both the constrained and linear peptide libraries. The design ofthe 14-mer allowed for some amino acid variation at each of theremaining 9 positions, however, preference was given for a particularamino acid at each of these positions. Analysis of binding affinity ofthe newly isolated peptides for BLyS was evaluated by direct andindirect phage ELISA and fluorescence anisotropy.

BAML was designed on a 14-mer linear (non-constrained) template peptidesequence having fixed residues at positions 1 (Ala), 5 (Asp), 7 (Leu), 8(Thr), and 10 (Leu). The amino acid sequence of positions 3-14 in theBAML template most closely resembles a binding polypeptide isolated fromthe PhD 12 linear polypeptide library (see Table 7, supra). Residues atposition 1 (fixed Ala) and position 2 (variable) were included to extendthe length and presentation of the BLyS-binding sequence. Positions 5-8correspond to the DxLT motif found in peptide isolates from both theconstrained and linear peptide libraries (see Tables 1-8, supra). Sincehydrophobic amino acids (L, M, I, A, and G) were found at position 10 in85% of the original isolates, a Leu residue, occurring in 42% of theisolates, was fixed at that position in the BAML template peptide. TABLE10 BAML template sequence (14-mer) amino acid position SEQ ID 1 2 3 4 56 7 8 9 10 11 12 13 14 NO: A n w y D s L T k L w l p d 184

Referring to Table 10, the upper case letters indicate the fixedresidues at positions 1, 5, 7, 8, and 10 of the template. Lower caseletters designate preferred amino acids at those positions, however thedesign of the variegated DNA template encoding the 14-mer allows forsome sequence variation at these positions.

Table 11 shows the design of the variegated DNA template used togenerate the BAML peptides. TABLE 11 BAML DNA template sequence (14-mer)codon position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 codons* GCT eez zjj zezGAT zqz CTT ACT eej CTC zjj qzz qqz jez*The sequence of codons is SEQ ID NO:185.

Referring to Table 11, the nucleotide coding sequences for the fixedamino acids in the BAML 14-mer template are shown in upper case letters.The letters “e”, “j”, “q”, and “z” in the variegated DNA template eachrepresent a particular mixture of nucleoside bases present in the inputdNTPs for each position:

-   -   j=79% guanine, 7% cytosine, 7% adenine, 7% thymine    -   q=7% guanine, 79% cytosine, 7% adenine, 7% thymine    -   e=7% guanine, 7% cytosine, 79% adenine, 7% thymine    -   z=7% guanine, 7% cytosine, 7% adenine, 79% thymine.        The codons of the DNA template were designed to skew the encoded        variable amino acid toward the preferred amino acid at each        position shown in SEQ ID NO:184 (Table 10, lower case). Later        sequencing of phage isolates showed that, at any particular        position, preferred residues occurred at a frequency of from 44%        to 70%.

Synthetic DNA sequences fitting the DNA template were amplified by largescale PCR. The amplified DNAs were restriction digested for insertioninto a M13 phage expression vector (MANP vector, Dyax Corp., Cambridge,Mass.), and vectors bearing the inserts were used to transform M13 phageby electroporation, to produce the BAML.

Recombinant phage were collected and purified by PEG precipitation andtitered. A total of 3.2×10¹³ PFU were amplified in BAML from 1.6×10⁹transformants.

Screening BAML

As outlined in Table 12 below, a two-step competition method, startingwith the original BAML library, was used over 4 rounds of screening toisolate the highest affinity BLyS-binding polypeptides from the BAML.Prior to screening, the amplified BAML was contacted with Seradynstreptavidin-coated magnetic beads (MG-SA, Seradyn, Indianapolis, Ind.),to remove bead- and streptavidin-binding phage.

For screening BAML, phage were incubated in solution with biotinylatedBLyS (b-BLyS) in 200 μl PBS, pH 7.4, Tween-20 (0.1%), to formphage/b-BLyS binding complexes. For the first competition step,unlabeled BLyS (1-2 μM) was added to the phage/b-BLyS binding complexmixture in solution and incubated for 1-20 hrs. (See Table 12.) Thephage/b-BLyS complexes remaining in solution after incubation withunlabeled BLyS were captured by brief (10 min. on rotator) incubationwith MG-SA streptavidin beads (50 μl). After capture of the phage/b-BLyScomplexes on streptavidin beads, the unbound fraction was removed andbeads were washed 15-20 times with 1 ml PBS-Tween 20 prior to the secondcompetition step. The phage/unlabeled BLyS complexes from the round 1competition step only, were collected and used as a fraction of theinput phage for the second round of screening along with thebead-captured phage/b-BLyS complexes, however, in each subsequent roundof screening only the bead-associated phage were collected after thefirst competition step for further screening, and the phage/unlabeledBLyS complexes were discarded.

For the second competition step, the competitor peptide was apolypeptide (DX221; SEQ ID NO:168) based on a BLyS-binding polypeptideisolated from the PhD 12 library in the initial screenings describedabove. The phage/b-BLyS complexes bound to streptavidin, collected afterthe first competition incubation step, were serially diluted with 50 μMDX221 BLyS-binding peptide (K_(D)=3 μM) in 300 μl PBS-Tween-20 (0.1%). Aseries of short incubations (3-4 per round, for 1 hour) of thephage/b-BLyS complexes with DX221 followed by a final incubation of fromovernight (O/N, for rounds 1, 2, and 4) to 3 days (for round 3). (SeeTable 12.) The second competition step in round 4 included an incubationwith 67 nM BLyS for 1 hour prior to incubation with DX221. Thestreptavidin bead-associated phage/b-BLyS binding complexes remainingafter the DX221 competition step in round 4 were collected for furtheranalysis. TABLE 12 BLyS affinity maturation library (BAML) screeningconditions First Second Competition Competition Screening InputIncubation Competitor Incubation Peptide Round phage¹ b-BLyS² Time (hrs)(BLyS) Time (hrs) Elutions 1 1.5 × 10¹¹ 100 nM 2 2 μM 1 50 μM DX221, 4 ×1 hr, then O/N 2   2 × 10¹⁰ 100 nM 1 1 μM 20 50 μM DX221, 3 × 1 hr, thenO/N 3 6.5 × 10¹⁰ 100 pM 16 1 μM 3 50 μM DX221, 4 × 1 hr, then 3 days 46.0 × 10¹⁰  10 pM 16 1 μM 2 67 nM BLyS, 1 hr; 50 μM DX221 + 67 nM BLyS 3× 1 hr, O/N, then add'l 4 hrs¹Input phage for round 1 was original BAML; for round 2 was amplifiedoutput phage from overnight (final) peptide elution and bead-associatedphage from round 1; for round 3 was amplified bead-associated outputphage from round 2; and for round 4 was amplified bead-associated outputphage from round 3.All amplified phage samples were pre-cleared on streptavidin beadsbefore incubation with biotin-BLyS in solution.²b-BLyS = biotinylated BLySELISA Analysis

Approximately four hundred BAML isolates from rounds 2, 3 and 4 of theabove screening were analyzed by direct and indirect phage ELISA assays.

For indirect phage ELISA, Immulon-2HB plates (Dynex Technologies, Inc.,Chantilly, Va.) were coated with 100 μl of 1 μg/ml Immunopurestreptavidin (Pierce, Rockford, Ill.) diluted in PBS. 100 μl of a seriesof 10-fold dilutions of b-BLyS (0-0.1 μg/ml in PBS) were immobilized inthe streptavidin-coated wells (1 hr, 37° C.). After washing, 1-25 μl ofovernight culture of E. coli infected with the individual phage plaqueswere added to the appropriate wells and incubated for 1 hour, followedby 10 washes with PBS-Tween-20. Anti-M13 antibody conjugated tohorseradish peroxidase (1:10,000 in PBS-Tween-20) was added to the wells(30 min., room temperature), the color reagent TMB was used and theplates read at OD 630 nm.

Individual phage isolates binding to immobilized BLyS were sequenced andthe sequences analyzed. The unique sequences of the BAML BLyS-binding14-mer display peptides are shown in Table 13.

Analysis of the peptides reveals a significant sequence “collapse”around one motif: W₃YDPLTKLWL₁₂ (SEQ ID NO:436) (subscripts indicateamino acid position in the 14-mer display peptide sequence). This mostnumerous core motif includes the four fixed residues from the originalBAML template, i.e., Asp (D) at position 5, Leu (L) at position 7, Thr(T) at position 8, and Leu (L) at position 10. In addition, 5 of the 6preferred residues from the original BAML template sequence wereincluded in this motif (see Table 10).

73% (143 of 197) of the round 4 isolates included this core motif (SEQID NO:436). Single residue substitutions within the 10-mer core motifcentered on positions 4 (Y→F) and 12 (L→F, I, or V), with thesubstitutions at position 12 being alternative hydrophobic residues forLeu.

For the three remaining variable positions (i.e., 2, 13, and 14),selection was not as stringent, although some preferences were apparent,being either built into the library or persisting through rounds ofselection. For example, in round 4 isolates, 51% included Asn atposition 2; 77% included Pro at position 13; and 32% included Asp atposition 14. The presence of Val (27%) or Glu (19%) at position 14 wasamong the most highly selected in the round 4 isolates, in comparison totheir theoretical proportion (4% each) at position 14 in BAML.

The sequences in Table 13 are grouped according to their degree ofdifference from the core sequence (SEQ ID NO:436). TABLE 13 Sequences ofBAML Phage Isolates (from Rounds 2, 3, 4) 14-mer amino acid position 1 23 4 5 6 7 8 9 10 11 12 13 14 SEQ ID NO: A n w y D s L T k L w l p dconsensus; 184 A N W Y D P L T K L W L P D 186 A N W Y D P L T K L W L PE 187 A N W Y D P L T K L W L P G 188 A N W Y D P L T K L W L P V 189 AN W Y D P L T K L W L P D 190 A N W Y D P L T K L W L P D 191 A N W Y DP L T K L W L P T 192 A N W Y D P L T K L W L P A 193 A N W Y D P L T KL W L P N 194 A N W Y D P L T K L W L V D 195 A N W Y D P L T K L W L HD 196 A N W Y D P L T K L W L T D 197 A N W Y D P L T K L W L P H 198 AN W Y D P L T K L W L T V 199 A N W Y D P L T K L W L L D 200 A N W Y DP L T K L W L L E 201 A N W Y D P L T K L W L H E 202 A N W Y D P L T KL W L P R 203 A N W Y D P L T K L W L A D 204 A N W Y D P L T K L W L PY 205 A N W Y D P L T K L W L P I 206 A N W Y D P L T K L W L I D 207 AN W Y D P L T K L W L R D 208 A Y W Y D P L T K L W L P D 209 A Y W Y DP L T K L W L L E 210 A Y W Y D P L T K L W L R V 211 A Y W Y D P L T KL W L P E 212 A Y W Y D P L T K L W L P V 213 A Y W Y D P L T K L W L HQ 214 A Y W Y D P L T K L W L P A 215 A Y W Y D P L T K L W L R V 216 AY W Y D P L T K L W L P G 217 A Y W Y D P L T K L W L R Y 218 A Y W Y DP L T K L W L P Y 219 A Y W Y D P L T K L W L L Y 220 A Y W Y D P L T KL W L R D 221 A Y W Y D P L T K L W L P V 222 A Y W Y D P L T K L W L LG 223 A Y W Y D P L T K L W L T H 224 A Y W Y D P L T K L W L P T 225 AY W Y D P L T K L W L L V 226 A Y W Y D P L T K L W L Y Y 227 A Y W Y DP L T K L W L S D 228 A S W Y D P L T K L W L P A 229 A S W Y D P L T KL W L H D 230 A S W Y D P L T K L W L P G 231 A S W Y D P L T K L W L PQ 232 A S W Y D P L T K L W L P Y 233 A S W Y D P L T K L W L P H 234 AS W Y D P L T K L W L P V 235 A S W Y D P L T K L W L P I 236 A S W Y DP L T K L W L P E 237 A F W Y D P L T K L W L R V 238 A F W Y D P L T KL W L P E 239 A F W Y D P L T K L W L L E 240 A F W Y D P L T K L W L PV 241 A I W Y D P L T K L W L P E 242 A I W Y D P L T K L W L P D 243 AI W Y D P L T K L W L H D 244 A I W Y D P L T K L W L T D 245 A I W Y DP L T K L W L P F 246 A I W Y D P L T K L W L L D 247 A I W Y D P L T KL W L P R 248 A I W Y D P L T K L W L P A 249 A I W Y D P L T K L W L TA 250 A I W Y D P L T K L W L A V 251 A I W Y D P L T K L W L P G 252 AI W Y D P L T K L W L R V 253 A I W Y D P L T K L W L P H 254 A I W Y DP L T K L W L R E 255 A I W Y D P L T K L W L S D 256 A T W Y D P L T KL W L S D 257 A T W Y D P L T K L W L P A 258 A T W Y D P L T K L W L AD 259 A T W Y D P L T K L W L T S 260 A T W Y D P L T K L W L P G 261 AT W Y D P L T K L W L P Y 262 A T W Y D P L T K L W L S G 263 A T W Y DP L T K L W L P V 264 A D W Y D P L T K L W L P V 265 A D W Y D P L T KL W L P K 266 A D W Y D P L T K L W L P D 267 A D W Y D P L T K L W L PE 268 A D W Y D P L T K L W L H Q 269 A E W Y D P L T K L W L R Q 270 AE W Y D P L T K L W L P D 271 A E W Y D P L T K L W L P Y 272 A L W Y DP L T K L W L P A 273 A L W Y D P L T K L W L P D 274 A L W Y D P L T KL W L R G 275 A L W Y D P L T K L W L L G 276 A M W Y D P L T K L W L PA 277 A M W Y D P L T K L W L Q V 278 A M W Y D P L T K L W L L G 279 AA W Y D P L T K L W L P D 280 A A W Y D P L T K L W L A D 281 A A W Y DP L T K L W L L D 282 A H W Y D P L T K L W L T D 283 A H W Y D P L T KL W L P V 284 A H W Y D P L T K L W L H D 285 A H W Y D P L T K L W L PD 286 A P W Y D P L T K L W L H D 287 A P W Y D P L T K L W L P V 288 AQ W Y D P L T K L W L P Y 289 A Q W Y D P L T K L W L P R 290 A Q W Y DP L T K L W L P D 291 A K W Y D P L T K L W L P V 292 A K W Y D P L T KL W L P V 293 A K W Y D P L T K L W L N G 294 A K W Y D P L T K L W L PA 295 A W W Y D P L T K L W L T D 296 A V W Y D P L T K L W L P A 297 AY E Y D P L T K L W L L Y 298 A T K Y D P L T K L W L P D 299 A T L Y DP L T K L W L P G 300 A I R Y D P L T K L W L P Y 301 A E R Y D P L T KL W L P H 302 A D R Y D P L T K L W L P Q 303 A N S Y D P L T K L W L PE 304 A I L Y D P L T K L W L P D 305 A N W F D P L T K L W L P Q 306 AN W F D P L T K L W L P V 307 A N W F D P L T K L W L T D 308 A N W F DP L T K L W L P D 309 A N W F D P L T K L W L P G 310 A N W F D P L T KL W L P E 311 A N W F D P L T K L W L P A 312 A N W F D P L T K L W L PN 313 A N W F D P L T K L W L S E 314 A N W F D P L T K L W L H D 315 AN W F D P L T K L W L V D 316 A Y W F D P L T K L W L P D 317 A Y W F DP L T K L W L P V 318 A Y W F D P L T K L W L P A 319 A Q W F D P L T KL W L P D 320 A H W F D P L T K L W L P D 321 A T W F D P L T K L W L PV 322 A Y W Y D S L T K L W L P V 323 A Y W Y D S L T K L W L H D 324 AN W Y D S L T K L W I P D 325 A N W Y D S L T K L W L P V 326 A N W Y DS L T K L W L P D 327 A N W Y D S L T K L W L A D 328 A N W Y D S L T KL W L P A 329 A N W Y D S L T K L W L Y E 330 A G W Y D S L T K L W L PD 331 A V W Y D S L T K L W L T D 332 A N W Y D A L T K L W L P V 333 AY W Y D T L T K L W L P N 334 A F W Y D P L T N L W L L E 335 A Y W Y DP L T G L W L L V 336 A Y W Y D P L T G L W L L Y 337 A Y W Y D P L T GL W L R V 338 A Y W Y D P L T E L W L R L 339 A M W Y D P L T K L S L PD 340 A Y W Y D P L T K L S L L V 341 A I W Y D P L T K L S L T V 342 AI W Y D P L T K L S L L V 343 A D W Y D P L T K L S L L L 344 A Y W Y DP L T K L R L L E 345 A D W Y D P L T K L R L L V 346 A D W Y D P L T KL R L I V 347 A I W Y D P L T K L Y L P D 348 A I W Y D P L T K L G L LV 349 A N W Y D P L T K L T L L V 350 A N W Y D P L T K L L L P N 351 AS W Y D P L T K L W F P D 352 A N W Y D P L T K L W F P D 353 A N W Y DP L T K L W F S D 354 A S W Y D P L T K L W F P V 355 A D W Y D P L T KL W F P V 356 A S W Y D P L T K L W F P K 357 A K W Y D P L T K L W F PD 358 A S W Y D P L T K L W F L E 359 A N W Y D P L T K L W F P A 360 AT W Y D P L T K L W F P D 361 A I W Y D P L T K L W F P E 362 A I W Y DP L T K L W F P D 363 A I W Y D P L T K L W F P G 364 A Y W Y D P L T KL W F P H 365 A N W Y D P L T K L W F P V 366 A Y W Y D P L T K L W F PD 367 A G W Y D P L T K L W F P D 368 A I W Y D P L T K L W F P T 369 AK W Y D P L T K L W F P A 370 A Y W Y D P L T K L W F F D 371 A N W Y DP L T K L W F A D 372 A N W Y D P L T K L W F P Y 373 A D W Y D P L T KL W F R D 374 A N W Y D P L T K L W V P D 375 A D W Y D P L T K L W V PA 376 A N W Y D P L T K L W V P N 377 A N W Y D P L T K L W V P E 378 AN W Y D P L T K L W V P Q 379 A E W Y D P L T K L W V P K 380 A Q W Y DP L T K L W V P V 381 A N W Y D P L T K L W V P Y 382 A L W Y D P L T KL W V P Y 383 A N W Y D P L T K L W V P G 384 A S W Y D P L T K L W I PY 385 A D W Y D P L T K L W I P G 386 A N W Y D P L T K L W I P Y 387 AK W Y D P L T K L W I P Y 388 A I W Y D P L T K L W I P N 389 A T W Y DP L T K L W I P Q 390 A S W Y D P L T N L W V P D 391 A Y E Y D P L T NL W L L Y 392 A Y W Y D P L T N L S L L V 393 A Y W Y D P L T K L S I LE 394 A N W Y D S L T K L W I P Y 395 A H W F D P L T Q L K I R V 396 AY W C D P L T K L C I L E 397 A N S Y D P L T K L W F P Y 398 A N L Y DP L T K L W V P Y 399 A N W Y D A L T K L W L H D 400 A N W Y D S L T KL W F P D 401 A T S Y D S L T K L W L P A 402 A C W Y D S L T K L C H RE 403 A I G N D P L T K L W I P Y 404 A N W Q D C L T K L C L A G 405 AY W F D P L T N L W L L E 406 A Y W Y D P L T N L S L L V 407 A N C F DS L T R L W L C D 408 A C A Y D A L T K L C L P A 409 A N W Y D P L T NL S L L L 410 A Y W Y D P L T Q L S L L V 411 A Y R Y D A L T G L W L LY 412 A Y W N D P L T K L K L R L 413 A Y W Y D P L T Q L S L L V 414 AY R Y D A L T G L W L L Y 415 A Y R Y D S L T N L W L L Y 416 A Y W Y DP L T K L S I L E 417 A S C Y D P L T K L C F P V 418 A F W F D P L T GL W L L E 419 A H W Y D P L T K L S I R V 420 A P W Y D S L T K L W F PS 421 A N C Y D T L T K L W L T C 422 A N W Y D S L T K L S L P D 423 AY A Y D F L T Q L S L P D 424 A F R Y D S L T G L W L R Y 425 A N C Y DS L T K L W L P C 426 A N G Y D L L T N L S V S D 427 A N W Y D P L T RL W I P V 428 A L K F D Y L T K L W L P D 429 A Y R Y D S L T K L W L PG 430 A Y C Y D S L T K L W I P D 431 A S W E D S L T K L W L S K 432 AY W Y D S L T G L S L L V 433 A Y W Y D P L T Y L R L R V 434 A K C Y DS L T N L W L C D 435

Nearly all of the ELISA signals of the BAML isolates were higher thanthose isolated in the initial screen (see Example 1). For comparison,peptide 453-01-B07 (SEQ ID NO:31) (K_(D)=700 nM) was used as a reference(positive control). Negative control MAEX (M13 phage with no insert) didnot bind b-BLyS at any concentration tested.

For direct phage ELISA, the signal measured is a reflection of theability of a set number of phage to bind to various concentrations ofb-BLyS. Peptides tested by the direct phage ELISA assay were chosenbased on high affinity for BLyS as determined in the indirect phageELISA assay. For this assay, Immulon-2HB plates were coated with 0 or100 ng anti-Fd antibody (Sigma, St. Louis, Mo.). After washing(PBS-Tween-20), phage dilutions were added to saturate the availableantibody and incubated for 1 hour, washed, then incubated with 100 μl of10-fold dilutions of b-BLyS (0-1 μg/ml) for 1 hour at room temperature.Streptavidin-HRP (1:1000 in PBS-tween-20; Endogen, Woburn, Mass.) wasadded to the wells and incubated for 1 hour, developed using TMB andreading at OD 630 mm.

Determination of BAML Peptide K_(D) by Fluoresence Anisotropy.

Several peptides containing the 10-mer core structural motif orsingle-position variants of that motif identified by sequence analysiswere synthesized with a short Gly-Gly-Lys linker sequence and theC-terminal lysine was labeled with fluorescein. These peptides, shown inTable 14, below, were synthesized by solid phase synthesis fordetermination of dissociation constant with respect to BLyS. The DX815and DX876 polypeptides were derived from DX814 (SEQ ID NO:186) bydeletion of two N-terminal amino acids or the two amino acids N-terminaland C-terminal to the core peptide at (positions 3-12). DX816, DX817,DX819, and DX822 correspond to other BAML isolates (SEQ ID NOs:189, 309,353, 327, respectively). DX818 corresponds to isolate SEQ ID NO:340,except that Asn has been substituted for Met at position 2. The K_(D) ofseveral BLyS binding BAML peptides was determined by fluorescenceanisotropy, performed as previously described. The sequence of DX822without the -GGK linker (see SEQ ID NO:327) matches the BAML templatesequence (see Table 10). The BAML consensus sequence found in DX822resulted in a more than 10-fold improvement in binding affinity forBLyS, as compared to one of the highest affinity binders isolated in theinitial screen (453-01-B07, SEQ ID NO:31). TABLE 14 DissociationConstants of Synthetic BLyS-binding Polypeptides SEQ ID Peptide SequenceNO: K_(d) (nM) DX814 Ac-ANWYDPLTKLWLPDGGK-fitc 437 26 ± 7  DX815Ac-WYDPLTKLWLPDGGK-fitc 438 31 ± 13 DX876 Ac-WYDPLTKLWLGGK-fitc 439 171± 90  DX816 Ac-ANWYDPLTKLWLPVGGK-fitc 440 44 ± 15 DX817Ac-ANWFDPLTKLWLPDGGK-fitc 441 32 ± 26 DX818 Ac-ANWYDPLTKLSLPDGGK-fitc442 342 ± 108 DX819 Ac-ANWYDPLTKLWFPDGGK-fitc 443 69 ± 38 DX822Ac-ANWYDSLTKLWLPDGGK-fitc 444 79 ± 54

Analysis of the BAML isolates revealed a lack of sequence conservationat position 2 (varied in the BAML template, see Table 10). To examinewhether the N-terminal residues at positions 1 and 2 in the BAMLsequence were necessary for binding to BLyS, a truncated version ofDX814 comprising only residues 3-14 (DX815; see Table 14) wassynthesized and analyzed by fluorescence anisotropy. The K_(D) for DX815was indistinguishable from that of DX814, suggesting that residues 1-2are not required for high affinity binding to BLyS. Further truncationof DX814 to the minimal core (residues 1-10, DX876) increased the K_(D)to 171 nM, indicating a contribution from Pro at position 13 and/or Aspat position 14 of the 14-mer to high affinity BLyS binding. Substitutionof Val in DX816 at that position had little effect on the K_(D) (seeTable 14). In comparing the BLyS-binding polypeptide DX221(Ac-WTDSLTGLWFPDGGPGPEGGGK; K_(D)=3 μM; SEQ ID NO:168) with the BAMLpeptide closest in sequence (DX819, Ac-ANWYDPLTKLWFPDGGK; K_(D)=69 nM;SEQ ID NO:443), differences are seen at three positions 4 (T→Y), 6(S→P), and 9 (G→K), indicating the contribution of these residues inbinding affinity.

The synthesized BAML peptides exhibited K_(D) values in the lownanomolar range, two orders of magnitude lower than primaryisolate-derived peptides (see Example 1). Phenylalanine substitutions(F₄→Y₄; F₁₂→L₁₂; Table 14) were the most common minor variations to thecore sequence and these changes failed to significantly affect thedissociation constants of the synthesized peptides. A change at position11 (W₁₁→S₁₁; DX818), however, resulted in an approximately 10-folddecrease in affinity compared to DX814.

Following the foregoing description, the characteristics important forusing various affinity binding polypeptides for targeting of BLyS orBLyS-like polypeptides (BLyS target protein) in vitro or in vivo can beappreciated. Additional binding polypeptide uses of the invention andalternative methods adapted to a particular use will be evident fromstudying the foregoing description. For instance, any spacer or linkersequences associated with BLyS binding polypeptides discussed above maybe removed or substituted to yield additional BLyS binding polypeptidesfor use in the methods of this invention. All such embodiments andobvious alternatives are intended to be within the scope of thisinvention, as defined by the claims that follow.

Publications referred to above are hereby incorporated by reference.

1. A method of treating, preventing or ameliorating a disease ordisorder associated with aberrant B Lymphocyte Stimulator (BLyS) or BLySreceptor expression or activity, comprising administering to an animalin which such treatment, prevention or amelioration is desired, a BLySbinding polypeptide in an amount effective to treat, prevent orameliorate the disease or disorder.
 2. The method of claim 1, whereinthe disease or disorder is an immune system disease or disorder.
 3. Themethod of claim 2, wherein the immune system disease or disorder is anautoimmune disease or disorder.
 4. The method of claim 2, wherein theimmune system disease or disorder is an immunodeficiency.
 5. The methodof claim 3, wherein the autoimmune disease or disorder is lupus.
 6. Themethod of claim 1, wherein the disease or disorder is glomerularnephritis.
 7. The method of claim 2, wherein the immune system diseaseor disorder is rheumatoid arthritis, multiple sclerosis,hypogammaglobulinemia or hypergammaglobulinemia.
 8. The method of claim2 wherein the immune system disease or disorder is graft vs. hostdisease.
 9. The method of claim 2, wherein the immune system disease ordisorder is a proliferative disease or disorder.
 10. The method of claim9, wherein the proliferative disorder is cancer.
 11. The method of claim1, wherein the disease or disorder is an infectious disease or disorder.12-68. (canceled)
 69. The method according to claim 1, wherein the BLySbinding polypeptide comprises an amino acid sequence selected from thegroup consisting of: (1) Asp-Xaa-Leu-Thr, (SEQ ID NO:446)

wherein Xaa is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Proor Ser); (2) X₁-X₂-X₃-Cys-X₅-Phe-X₇-Trp-Glu-Cys-X₁₁-X₁₂-X₁₃, (SEQ IDNO:1)

wherein X₁ is Ala, Asn, Lys, or Ser; X₂ is Ala, Glu, Met, Ser, or Val;X₃ is Ala, Asn, Lys, or Pro (preferably Lys); X₅ is Phe, Trp, or Tyr(preferably Tyr); X₇ is Pro or Tyr (preferably Pro); X₁₁ is Ala, Gln,His, Phe, or Val; X₁₂ is Asn, Gln, Gly, His, Ser, or Val; and X₁₃ isAla, Asn, Gly, Ile, Pro, or Ser; (3)X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-Cys-X₁₂-X₁₃-X₁₄, (SEQ ID NO:2)

wherein X₁ is Ala, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, Val, or is absent; X₂ is Ala, Asn, Asp, GlnGly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X₃is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr, or Val (preferably Asp); X₅ is Asp, Ile, Leu, or Tyr(preferably Asp or Leu); X₆ is Arg, Asp, Glu, His, Ile, Leu, Lys, Phe,Pro, Tyr, or Val (preferably Glu or Leu); X₇ is His, Leu, Lys, or Phe(preferably His or Leu); X₈ is Leu, Pro, or Thr (preferably Thr or Pro);X₉ is Arg, Asn, Gly, His, Ile, Lys, Met, or Trp (preferably Lys); X₁₀ isAla, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Tip, Tyr, or Val; X₁₂is Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Trp, Tyr, or Val; X₁₃ isAla, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val; and X₁₄ is Ala, Arg, Asn, Asp, Gln, Glu, Gly,His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, Val, or is absent; (4)X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys-X₁₃-X₁₄-X₁₅, (SEQ ID NO:3)

wherein X₁ is Ala, Arg, Asn, Asp, Leu, Lys, Phe, Pro, Ser, or Thr; X₂ isAsn, Asp, Gln, His, Ile, Lys, Pro, Thr, or Trp; X₃ is Ala, Arg, Asn,Gln, Glu, His, Phe, Pro, or Thr (preferably Ala); X₅ is Asn, Asp, Pro,Ser, or Thr (preferably Asp); X₆ is Arg, Asp, Ile, Leu, Met, Pro, or Val(preferably Ile); X₇ is Ala, Ile, Leu, Pro, Thr, or Val (preferably Valor Leu); X₈ is Asn, His, Ile, Leu, Lys, Phe, or Thr (preferably Thr); X₉is Asn, Glu, Gly, His, Leu, Lys, Met, Pro, or Thr (preferably Leu); X₁₀is Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Ser, or Trp; X₁₁ isArg, Glu, Gly, Lys, Phe, Ser, Trp, or Tyr (preferably Ser); X₁₃ is Gln,Glu, Ile, Leu, Phe, Pro, Ser, Tyr, or Val (preferably Val); X₁₄ is Asn,Gly, Ile, Phe, Pro, Thr, Trp, or Tyr; and X₁₅ is Asn, Asp, Glu, Leu,Lys, Met, Pro, or Thr (preferably Glu or Pro); (5)X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-Cys-X₁₄-X₁₅-X₁₆, (SEQ ID NO:4)

wherein X₁ is Asn, Asp, His, Leu, Phe, Pro, Ser, Tyr, or is absent(preferably Ser); X₂ is Arg, Asn, Asp, His, Phe, Ser, or Trp (preferablyArg); X₃ is Asn, Asp, Leu, Pro, Ser, or Val (preferably Asn or Asp); X₅is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₆ is His, Ile, Leu,Met, Phe, Pro, Trp, or Tyr; X₇ is Asp, His, Leu, or Ser (preferablyAsp); X₈ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr (preferably Glu orPro); X₉ is Ala, Arg, Asn, or Leu (preferably Leu); X₁₀ is Ile, Leu,Met, Pro, Ser, or Thr (preferably Thr); X₁₁ is Ala, Arg, Asn, Gly, His,Lys, Ser, or Tyr; X₁₂ is Ala, Arg, Asn, Gln, Leu, Met, Ser, Trp, Tyr, orVal; X₁₄ is Asp, Gly, Leu, Phe, Tyr, or Val (preferably Leu); X₁₅ isAsn, His, Leu, Pro, or Tyr (preferably His, Leu or Pro); and X₁₆ is Asn,Asp, His, Phe, Ser, or Tyr, (preferably Asp or Ser); (6)X₁-X₂-X₃-Cys-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-Cys-X₁₆-X₁₇-X₁₈, (SEQ IDNO:5)

wherein X₁ is Arg, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp, Tyr, or isabsent (preferably Arg); X₂ is Ala, Arg, Asn, Asp, Gly, Pro, Ser, or isabsent (preferably Asn, Asp, Gly, or Pro); X₃ is Arg, Asn, Gln, Glu,Gly, Lys, Met, Pro, Trp or Val (preferably Gly or Met); X₅ is Arg, Asn,Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val preferably Trp, Tyr, orVal); X₆ is Arg, Asp, Gln, Gly, Ile, Lys, Phe, Thr, Trp or Tyr(preferably Asp); X₇ is Ala, Arg, Asp, Glu, Gly, Leu, Ser, or Tyr(preferably Asp); X₈ is Asp, Gln, Glu, Leu, Met, Phe, Pro, Ser, or Tyr(preferably Leu); X₉ is Asp, Leu, Pro, Thr, or Val (preferably Leu orThr); X₁₀ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp or Tyr(preferably Lys or Thr); X₁₁ is Ala, Arg, Asn, Gln, Glu, His, Leu, Lys,Met, or Thr (preferably Arg or Leu); X₁₂ is Ala, Asn, Gln, Gly, Leu,Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp); X₁₃ is Ala,Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferably Met orPhe); X₁₄ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Tyr,or Val (preferably Val); X₁₆ is Arg, Asp, Gly, His, Lys, Met, Phe, Pro,Ser, or Tip (preferably Met); X₁₇ is Arg, Asn, Asp, Gly, His, Phe, Pro,Ser, Trp or Tyr, (preferably Arg, His, or Tyr); and X₁₈ is Ala, Arg,Asn, Asp, His, Leu, Phe, or Tip (preferably His or Asn); (7)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂, (SEQ ID NO:6)

wherein X₁ is Ala, Arg, Gly, His, Leu, Lys, Met, Phe, Trp, Tyr, or Val(preferably Gly, Tyr, or Val); X₂ is Ala, Arg, Gln, His, Ile, Leu, Phe,Thr, Trp, or Tyr (preferably His or Tyr); X₃ is Ala, Asp, Lys, Phe, Thr,Trp or Tyr (preferably Asp or Tyr); X₄ is Arg, Asp, Gln, Lys, Met, Phe,Pro, Ser, Tyr, or Val (preferably Asp or Gln); X₅ is Asp, Leu, Lys, Phe,Pro, Ser, or Val (preferably Leu or Ser); X₆ is His, Ile, Leu, Pro, Ser,or Thr (preferably Leu or Thr); X₇ is Arg, Gly, His, Leu, Lys, Met, orThr (preferably Lys or Thr); X₈ is Ala, Arg, Asn, Ile, Leu, Lys, Met, orThr (preferably Leu or Lys); X₉ is Ala, Asn, Arg, Asp, Glu, Gly, His,Leu, Met, Ser, Trp, Tyr, or Val (preferably Met or Ser); X₁₀ is Ile,Leu, Phe, Ser, Thr, Trp, Tyr, or Val (preferably Thr or Leu); X₁₁ isAla, Arg, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Trp, Tyr, or Val(preferably Pro or Thr); and X₁₂ is Arg, Asp, His, Leu, Lys, Met, Phe,Pro, Ser, Trp, Tyr, or Val (preferably Arg or Pro); (8)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃, (SEQ ID NO:7)

wherein X₁ is Asp, Gin, Glu, Gly, His, Lys, Met, or Trp (preferably Gluor Lys); X₂ is Arg, Gin, His, Ile, Leu, or Pro (preferably His or Pro);X₃ is Asp, Gly, Ile, Lys, Thr, Tyr or Val (preferably Tyr); X₄ is Asn,Asp, Gin, Glu, Met, Pro, Ser, or Tyr (preferably Asp or Gin); X₅ is Asn,Asp, His, Ile, Leu, Met, Pro, Thr or Val (preferably Asn or Thr); X₆ isAsp, Glu, His, Leu, Lys, Pro, or Val (preferably Asp or Pro); X₇ is Arg,Asn, Gin, His, Ile, Leu, Met, Pro, or Thr (preferably Ile or Pro); X₈ isGin, Gly, His, Leu, Met, Ser, or Thr (preferably Leu or Thr); X₉ is Asn,Gin, Gly, His, Leu, Lys, Ser, or Thr (preferably Lys); X₁₀ is Ala, Gly,Ile, Leu, Lys, Met, or Phe (preferably Gly or Met); X₁₁ is Ala, Glu,His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, or Val (preferably Ala or Thr);X₁₂ is Arg, Gin, Glu, Gly, His, Ile, Lys, Tyr, or Val (preferably Arg orHis); and X₁₃ is Arg, Asn, Glu, His, Ile, Ser, Thr, Trp, or Val(preferably His); (9) Cys-X₂-Phe-X₄-Trp-Glu-Cys, (SEQ ID NO:8)

wherein X₂ is Phe, Trp, or Tyr (preferably Tyr); and X₄ is Pro or Tyr(preferably Pro); (10) Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys, (SEQ ID NO:9)

wherein X₂ is Asp, Ile, Leu, or Tyr (preferably Asp or Leu); X₃ is Arg,Asp, Glu, His, Ile, Leu, Lys, Phe, Pro, Tyr, or Val (preferably Glu orLeu); X₄ is His, Leu, Lys, or Phe (preferably His or Leu); X₅ is Leu,Pro, or Thr (preferably Thr or Pro); X₆ is Arg, Asn, Gly, His, Ile, Lys,Met, or Trp (preferably Lys); and X₇ is Ala, Asn, Gin, Glu, Gly, His,Ile, Leu, Met, Phe, Ser, Trp, Tyr, or Val; (11)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Cys, (SEQ ID NO:10)

wherein X₂ is Asn, Asp, Pro, Ser, or Thr (preferably Asp); X₃ is Arg,Asp, Ile, Leu, Met, Pro, or Val (preferably Ile); X₄ is Ala, Ile, Leu,Pro, Thr, or Val (preferably Val or Leu); X₅ is Asn, His, Ile, Leu, Lys,Phe, or Thr (preferably Thr); X₆ is Asn, Glu, Gly, His, Leu, Lys, Met,Pro, or Thr (preferably Leu); X₇ is Arg, Asn, Asp, Gln, Glu, Gly, Ile,Lys, Met, Pro, Ser, or Trp; X₈ is Arg, Glu, Gly, Lys, Phe, Ser, Trp, orTyr (preferably Ser); (SEQ ID NO:11) (12)Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Cys,

wherein X₂ is Asp, Gln, His, Ile, Leu, Lys, Met, Phe, or Thr; X₃ is His,Ile, Leu, Met, Phe, Pro, Trp, or Tyr; X₄ is Asp, His, Leu, or Ser(preferably Asp); X₅ is Ala, Arg, Asp, Glu, Leu, Phe, Pro, or Thr(preferably Glu or Pro); X₆ is Ala, Arg, Asn, or Leu (preferably Leu);X₇ is Ile, Leu, Met, Pro, Ser, or Thr (preferably Thr); X₈ is Ala, Arg,Asn, Gly, His, Lys, Ser, or Tyr; X₉ is Ala, Arg, Asn, Gln, Leu, Met,Ser, Trp, Tyr, or Val; (13) Cys-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-Cys,(SEQ ID NO:12)

wherein X₂ is Arg, Asn, Gln, Glu, His, Leu, Phe, Pro, Trp, Tyr, or Val(preferably Trp, Tyr, or Val); X₃ is Arg, Asp, Gln, Gly, Ile, Lys, Phe,Thr, Trp or Tyr (preferably Asp); X₄ is Ala, Arg, Asp, Glu, Gly, Leu,Ser, or Tyr (preferably Asp); X₅ is Asp, Gln, Glu, Leu, Met, Phe, Pro,Ser, or Tyr (preferably Leu); X₆ is Asp, Leu, Pro, Thr, or Val(preferably Leu or Thr); X₇ is Arg, Gln, His, Ile, Leu, Lys, Met, Phe,Thr, Trp or Tyr (preferably Lys or Thr); X₈ is Ala, Arg, Asn, Gln, Glu,His, Leu, Lys, Met, or Thr (preferably Arg or Leu); X₉ is Ala, Asn, Gln,Gly, Leu, Lys, Phe, Pro, Thr, Trp, or Tyr (preferably Thr or Trp); X₁₀is Ala, Arg, Gln, His, Lys, Met, Phe, Pro, Thr, Trp, or Tyr (preferablyMet or Phe); X₁₁ is Arg, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser,Thr, Tyr, or Val (preferably Val); (14)Ala-X₂-X₃-X₄-Asp-X₆-Leu-Thr-X₉-Leu-X₁₁-X₁₂-X₁₃-X₁₄, (SEQ ID NO:447)

wherein X₂ is Asn, Ser, Tyr, Asp, Phe, Ile, Gln, His, Pro, Lys, Leu,Met, Thr, Val, Glu, Ala, Gly, Cys, or Trp (i.e., any amino acid exceptArg; preferably Asn); X₃ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, orSer (preferably Trp); X₄ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr); X₆is Pro, Ser, Thr, Phe, Leu, Tyr, Cys, or Ala (preferably Pro or Ser); X₉is Lys, Asn, Gln, Gly, or Arg (preferably Lys); X₁₁ is Trp, Ser, Thr,Arg, Cys, Tyr, or Lys (preferably Trp); X₁₂ is Leu, Phe, Val, Ile, orHis (preferably Leu); X₁₃ is Pro, Leu, His, Ser, Arg, Asn, Gln, Thr,Val, Ala, Cys, Ile, Phe, or Tyr (i.e., not Asp, Glu, Gly, Lys, Met, orTrp; preferably Pro); and X₁₄ is Asp, Glu, Asn, Val, His, Gln, Arg, Gly,Ser, Tyr, Ala, Cys, Lys, Ile, Thr or Leu (i.e., not Phe, Met, Pro, orTrp; preferably Asp); and, (15) X₁-X₂-Asp-X₄-Leu-Thr-X₇-Leu-X₉-X₁₀, (SEQID NO:448)

wherein X₁ is Trp, Glu, Lys, Cys, Leu, Ala, Arg, Gly, or Ser (preferablyTrp); X₂ is Tyr, Phe, Glu, Cys, Asn (preferably Tyr); X₄ is Pro, Ser,Thr, Phe, Leu, Tyr, Cys, or Ala preferably Pro or Ser); X₇ is Lys, Asn,Gln, Gly, or Arg (preferably Lys); X₉ is Trp, Ser, Thr, Arg, Cys, Tyr,or Lys (preferably Trp); and X₁₀ is Leu, Phe, Val, Ile, or His(preferably Leu).
 70. The method according to claim 69, wherein the BLySbinding polypeptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 20-168 and 186-435, as depicted inTables 1-8 and
 13. 71. The method according to claim 69, wherein theBLyS binding polypeptide comprises an amino acid sequence selected fromthe group consisting of:Ala-Gly-Lys-Glu-Pro-Cys-Tyr-Phe-Tyr-Trp-Glu-Cys-Ala-Val-Ser-Gly (SEQ IDNO: 450);Ala-Gly-Val-Pro-Phe-Cys-Asp-Leu-Leu-Thr-Lys-His-Cys-Phe-Glu-Ala-Gly (SEQID NO: 451);Gly-Ser-Ser-Arg-Leu-Cys-His-Met-Asp-Glu-Leu-Thr-His-Val-Cys-Val-His-Phe-Ala-Pro(SEQ ID NO: 452);Gly-Asp-Gly-Gly-Asn-Cys-Tyr-Thr-Asp-Ser-Leu-Thr-Lys-Leu-His-Phe-Cys-Met-Gly-Asp-Glu(SEQ ID NO: 453);Gly-Tyr-Asp-Val-Leu-Thr-Lys-Leu-Tyr-Phe-Val-Pro-Gly-Gly (SEQ ID NO:454); Trp-Thr-Asp-Ser-Leu-Thr-Gly-Leu-Trp-Phe-Pro-Asp-Gly-Gly (SEQ IDNO: 455); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQID NO:186); Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ ID NO:456); Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu (SEQ ID NO: 457);Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Val (SEQ ID NO:189);Ala-Asn-Trp-Phe-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp (SEQ ID NO:309); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Ser-Leu-Pro-Asp (SEQ IDNO: 458); Ala-Asn-Trp-Tyr-Asp-Pro-Leu-Thr-Lys-Leu-Trp-Phe-Pro-Asp (SEQID NO: 353); Ala-Asn-Trp-Tyr-Asp-Ser-Leu-Thr-Lys-Leu-Trp-Leu-Pro-Asp(SEQ ID NO: 327).